[Endoscopic visualization of luminal organ and great vessels with three dimensional CT scanner. A new trial using volumetric CT]. Thirty cases examined by three dimensions CT scanner (3DCT) were reported. The observation of inner view using 3DCT were performed in 12 large vessels with vascular disorder, 10 pulmonary bronchus with lung cancer and 8 common bile ducts involved obstructive disease. In order to visualize interface of the lumen, a new software which was devekioed by HITACHI MEDICO Inc. and us was brought in. In all cases but one the inner view of the luminal organ was clearly demonstrated as 3D images and it was possible to judge some changes of luminal interface involved by the diseases. The 3DCT endoscopic image might be useful as a new endoscopic technique without fiberscopy.


Case management: within and beyond the walls. Results of this study demonstrated no significant differences in the frequency of the instrumental use of case management skills between nurses who graduated from the NAMFE program (NAMFE group) and those who were about to enter the program (comparison group); however, there was a significant difference (p < .0001 level) in the perceived preparation for performance of these skills between the two groups. Currently, few educational programs provide nurses with the essential knowledge and skills to function in the role of case manager; yet, the job market demands it of them. The program was able to fill the gap between education and service. Although the NAMFE course no longer is available for continuing education credit through the University of Kansas School of Nursing, the course currently is offered as an elective for senior level students and graduate students. Also, the curriculum materials and teaching strategies manuals, developed as part of this grant, and consultations regarding the use of these materials are available.


Diversity in nursing faculty hinges on diversity in nursing students. In this chapter, I attempted to explicate and highlight some of the interrelationships which exist in higher education between white male and female faculty members from a minority perspective. I discussed the effect of attitudes on the success of minority students and faculty in light of projected demographic changes. I hope that white female faculty members will become more cognizant of how negative attitudes impact upon both their own performance in higher education and also how the inculcation of these attitudes can be transferred to minority faculty members with devastating results. Change is difficult in all spheres of life, but without some modicum of change, life will go on as usual. Minority faculty members and students deserve at the very least a fair chance in the game we call higher education.


Distance education: turf and technology. Distance learning fits with the mission and strategic plan of the University of Texas at Arlington. We believe these educational opportunities in nursing are highly desirable. The Board of Nurse Examiners for the State of Texas has approved this project and the Texas Higher Education Coordinating Board has approved it as a pilot project. The school will continue evaluation and creative problem-solving in the use of distance education.


[Good experiences with telemedicine in the regional hospital of Tromsø]. The University Hospital of Tromsø and the Norwegian Telecom Research project "Telemedicine in North Norway" has used video conferences as a tool in making remote diagnosis in the medical areas: Pathology, diagnosis from microscope images; dermatology, endoscopy in otorhinolaryngology, echocardiology and radiology. The project has also developed a communication system between general physicians and the department of the clinical chemistry in which test results are transported electronically from the laboratory to the physicians. Experience to date suggests that the quality of telecommunication in diagnostic services is good and that access to such facilities is beneficial to the users.


Epidemiology of childhood diabetes in the Baltic area. Insulin-dependent diabetes mellitus (IDDM) is a chronic disease with unknown aetiology and therefore without effective measures for its prevention. Both genetic and environmental factors play a role in the development of the disease. Comparative studies between different populations of varying genetic and environmental backgrounds are needed for better understanding of the aetiology of IDDM.


[Insulin treatment--its physiological principle]. Insulin treatment based on the physiological principle not only results in better glucose homeostasis but also in improved quality of life for the patients. The frequency of metabolic events also seems to be controlled, particularly when the multiple injection system is used. However, a prerequisite is regular, preferably daily, blood sugar monitoring to enable the insulin dosage to be adapted according to food intake and exercise. Advances in our knowledge of the importance of the injection technique used may contribute to the optimisation of insulin treatment and thus of glucose homeostasis.


[Chronic inflammatory bowel disease].

[Chronic inflammatory bowel disease]. Chronic inflammatory bowel disease (IBD) encompasses the disease entities, ulcerative colitis (UC) and Crohn's disease (CD). An aetiologic agent has not yet been defined and the diagnosis is based, therefore, on the sum of clinical, paraclinical, radiologic, endoscopic and histopathologic features. In recent years pathogenetic studies have focused on immune mechanisms, transmissible infectious agents, the potential role of the normal intestinal flora, dietary factors, enzymatic alterations and genetic features, in addition to vascular, neuromotor, allergic and psychologic factors. The corner stones in medical therapy of IBD are still corticosteroids and sulphasalazine (SAZ). The new oral salicylates, which are analogues of SAZ or "slow release" preparations of 5-aminosalicylic acid (mesalazine), have provided a therapeutic progress, because they are tolerated better than SAZ. Immunosuppressive agents, such as azathioprine and 6-mercaptopurine, reduce the requirement for corticosteroids and are effective in refractory CD, but the effect is delayed up to several months. The therapeutic action of cyclosporine A is not sustained, but often associated with side effects. Metronidazole has a beneficial effect on perineal disease. The efficacy of antimycobacterial drugs, sodium-cromoglycate, lidocaine, clonidine and sucralfate has been reported only in optimistic case stories and small open trials. A diet, rich in omega-3-fatty acids, modifies leukotriene (LT) production, but its clinical efficacy is insufficient. The first anti-leukotriene-drug, zileuton, has recently been evaluated and a significant, although insufficient, clinical response was obtained by a 70 per cent inhibition of rectal LTB4 synthesis. Dietary therapy may be useful as an adjunct to treatment of local complications in CD.(ABSTRACT TRUNCATED AT 250 WORDS)


[Which factors cause type-1 diabetes?].

[Which factors cause type-1 diabetes?]. Type 1 diabetes is due to selective and irreversible destruction of pancreatic beta-cells via an autoimmune process. The pathogenetic course is dependent both upon genetic predisposition for the disease and upon one or more environmental factors--eg, viral infection, dietary factors, and exposure to toxins or mental stress.


[Can type-1 diabetes in children be prevented?]. Increased knowledge of the etiopathogenesis of Type 1 diabetes has focused great interest on the possibilities of preventing the disease. Type 1 diabetes is considered to be a chronic autoimmune disease characterized by gradual beta-cell destruction mediated by autoreactive T-lymphocytes during an asymptomatic prediabetic phase of varying duration. Both experimental and epidemiologic data indicate that nutritional cow milk exposure early in life may play a critical role in the initiation of beta-cell destruction. Accordingly a primary prevention study has been planned to test the hypothesis that dietary elimination of cow milk proteins over the first 9 months of life will decrease the subsequent risk of childhood type 1 diabetes in high risk infants. The possibility of identifying prediabetic individuals before decisive loss of beta-cell function by various islet cell-specific autoantibodies enables measures of secondary prevention in the prediabetic phase. There are indications from experimental and human studies that nicotinamide, a water-soluble group B vitamin, may be effective in preventing or delaying the presentation of diabetes. A European multicentre study will be initiated in the near future to explore whether oral nicotinamide can prevent or delay the clinical manifestation of Type 1 diabetes in high risk first degree relatives of diabetic children. We have to wait for the results of these intervention studies for years, and similarly other prevention strategies have to be tested in large-scale long-lasting clinical trials. Nevertheless, prevention of childhood diabetes may become a reality in the next century.


[Diabetic coma--an unnecessary death].

[Diabetic coma--an unnecessary death]. Diabetic ketoacidosis remains a significant cause of death in cases of insulin-dependent diabetes mellitus (IDDM). Among patients hospitalised for diabetic ketoacidosis, the death rate is 5-10 per cent, cardiovascular disease, infection, and ARDS (adult respiratory distress syndrome) being major contributory factors, whereas the degree of acidosis does not differ from that among survivors. Ketoacidosis is a major determinant of the two-fold higher mortality among the youngest age-groups of IDDM patients. The age-specific incidence of ketoacidosis among patients under 20 years of age is several time higher than that among patients over 50. Intensified insulin treatment, using multiple injections or insulin pumps, probably results in an increased risk of insulin deficiency owing to the smaller insulin depots. Thus, there is a need of intensified testing for ketonuria and improved education of patients, physicians and other health care personnel, in order to promote the prevention or rapid, effective treatment of diabetic ketoacidosis.


[Diabetic nephropathy].

[Diabetic nephropathy]. Diabetic nephropathy is a common complication in diabetes mellitus. In addition to the risk of renal failure, patients with established nephropathy are at increased risk of proliferative retinopathy and cardiovascular disease. As the earliest prodrome of nephropathy is microalbuminuria, albumin excretion needs to be monitored with a reliable method in all diabetics. In the event of microalbuminuria, diabetes treatment needs to be intensified to optimise metabolic regulation. Early institution of antihypertensive treatment is essential to avoid progression to clinical nephropathy.


[Insulin-dependent diabetes mellitus--improved prognosis and insurance outlook]. The marked improvement during recent years in prognosis for patients with insulin-dependent diabetes mellitus (IDDM), has also had actuarial consequences. In the article is described the development in prognosis over the past 60 years, based (inter alia) on findings in almost 3,000 IDDM patients with onset during the period, 1933-1972, and referred to the Niels Steensens Hospital in Denmark.


[Treatment of advanced anal cancer].

[Treatment of advanced anal cancer]. Anal carcinoma affects between 30 and 40 Norwegians every year. The disease is twice as frequent in women as in men. It is usually located in the anal canal only, and is cured by a combination treatment with chemotherapy and radiotherapy. Metastatic anal carcinoma has a poor prognosis. We present two such cases. 5-fluorouracil in combination with cisplatin or mitomycin C can give responses even in advanced cases. Radiotherapy may also be of value. In view of the difference between norwegian clinics in the treatment of anal carcinoma, there would appear to be a need of a Scandinavian or international study based on standardized treatment.


[Hospice care in general practice?].

[Hospice care in general practice?]. In a primary health care district in southern Sweden, a programme was launched to treat terminally ill cancer patients in their own homes, on the lines of the hospice mode. The quality of life of these patients has been found to be improved, and the cost to the community to be less than that of institutionalised hospital care. Other patient categories that might benefit from this form a care are those with AIDS, severe neurological diseases, or chronic cardiac or respiratory insufficiency.


[Lead and health].

[Lead and health]. Large sums are being invested in the USA at present with a view to reducing the exposure of risk groups to lead. The Centers for Disease Control (Atlanta) have recently issued new limits for lead concentrations in children, the Occupational Safety and Health Administration is preparing to issue new threshold limit values (TLVs) for airborne lead in the USA, and a national laboratory control system is being built up.


Questions concerning plasma cell leukaemia. The classical concept of plasma cell leukaemia (PCL) is reviewed. This disease invariably concerns myeloma, either in its terminal phase (secondary PCL) or particularly rapid and aggressive (primary PCL). A more precise definition of primary PCL would be given by the term leukaemic myelomatosis. Characterization of the membrane antigen CD 56, responsible for the cytoadhesion of malignant plasmocytes in the bone marrow, today offers a convincing pathogenic hypothesis to elucidate this affection which would now appear to be no more than a variant form of myeloma. Acute immunoblastic-plasmoblastic leukaemia and mature cell leukaemic lymphoma would seem to be pathologically related to PCL. Finally, the problems posed by the "enigmatic" plasmacytoid monocyte are discussed.


Progress and survival factors in acute non lymphocytic leukemia. A 15 year analysis. We analyzed a population of adults with acute non lymphocytic leukemia (ANLL) treated from 1972-1989 to identify prognostic factors and the influence of therapy over time. To 179 patients treated at Sunnybrook Medical Centre (SMC) were added 114 patients from published patient series from Princess Margaret Hospital (PMH) all treated during that period. All PMH and 98 SMC patients received one of three remission induction protocols: CAV (cyclophosphamide, cytosine arabinoside [ara-C], vincristine) 1973-1976 (n = 46); ACT (Adriamycin, ara-C +/- 6-thioguanine) 1976-1983 (n = 83); high dose ara-C and corticosteroids 1983-1987 (n = 83). The remainder either received supportive therapy only or minimally toxic therapy (e.g., low dose ara-C, 6-mercaptopurine) due to presenting complications such as advanced age, severe concurrent medical condition, or most recently at SMC only, mitoxanthrone and ara-C, and were excluded from analysis. Responses obtained at the two institutions were identical, no survival advantage of any particular protocol was seen. Overall median survival was 8.5 months for patients treated on protocol and 20 months for those entering complete remission. Patients treated with supportive or minimally toxic therapy (n = 76) had a median survival of less than two months. In multivariate analysis, the only important factors for survival were complete response to initial therapy; complete response to second induction therapy following either first relapse (n = 89) or lack of complete response to first therapy (n = 15); and normal cytogenetic analysis.(ABSTRACT TRUNCATED AT 250 WORDS)


Monitoring of erythropoiesis by serum transferrin receptor levels in a case of chronic lymphocytic leukaemia and pure red cell aplasia treated with ciclosporin. The authors present the case of a patient with chronic lymphocytic leukaemia complicated by pure red cell aplasia. Successful treatment with ciclosporin was facilitated by assay of serum transferrin receptor, which demonstrated a prompt and sustained response of marrow erythropoiesis to this therapy.


Serum and erythrocyte ferritin in regular blood donors. This study evaluated 217 regular blood donors, with the aim of measuring their iron stores and recording the influence of donation frequency. In the population studied, 10% of men and 15% of women showed reduced iron stores of the erythropoietic marrow (erythrocyte ferritin less than the normal range) and 64% of the population showed a latent deficiency in iron (serum ferritin values less than the reference values). An increase in donation frequency was followed by an important decrease in serum ferritin concentration. Blood haemoglobin levels were inferior to control limits in 4% of men and 15% of women, measurements of haemoglobin being the legal screening test performed at the time of donation. However, this test gives a late indication of an already established iron deficiency and should be replaced by the determination of serum ferritin in order to avoid the possible appearance of iron deficiency. Erythrocyte ferritin is another late indicator of iron depletion and hence is of little interest for the control of blood donors.


A complex pattern of antigen receptor gene rearrangements in a case of Philadelphia positive acute lymphoblastic leukaemia. The paper describes a case of Philadelphia (Ph) positive acute lymphoblastic leukaemia (ALL) presenting with high white cell count and central nervous system involvement. Immunophenotypically the case was characterized as common ALL. The t(9;22) abnormality corresponded to a rearrangement within the breakpoint cluster region gene, while antigen receptor gene studies showed multiple rearrangements of the immunoglobulin heavy chain gene (IGH) concomitant with a single rearrangement of the T cell receptor beta chain gene (TCR beta). We speculate that this case represents the neoplastic transformation of a stem cell, the Ph abnormality being involved in the early steps of transformation. It is conceivable that the IGH but not the TCR beta gene was accessible to recombination within the malignant clone, thus generating the multiple rearrangements observed. If this is the case, these findings would appear to be compatible with the hypothesis that antigen receptor gene rearrangements may be partly dependent on the accessibility of the corresponding genetic loci.


Hairy cell leukemia and factor VIII inhibitor: a case report. Acquired factor VIII inhibitors are usually described in hemophilia A, although some cases have been documented in chronic inflammatory diseases and malignant tumors. We report here the first case of a factor VIII inhibitor appearing in the course of hairy cell leukemia. Interferon therapy over a 5 month period led to complete remission of leukemia with parallel disappearance of the acquired factor VIII inhibitor.


Tuberculosis associated hemophagocytic syndrome complicated with severe bone marrow failure and disseminated intravascular coagulation. Tuberculosis associated hemophagocytic syndrome (HPS) has recently been recognized as a benign reactive histiocytic proliferation with marrow hemophagocytosis. To our knowledge, only five autopsy documented cases have previously been reported. We present here a unique case of the disorder complicated by severe bone marrow failure and disseminated intravascular coagulation. The possible mechanisms of these complications are discussed and it is concluded that the immunological disturbances usually occurring in miliary tuberculosis could play a role in the pathogenesis of HPS.


Identification of Ets-like lymphoid specific elements within the immunoglobulin heavy chain 3' enhancer. Recently we identified an additional enhancer in the 3' end of the immunoglobulin heavy chain (IgH) locus. To identify individual regulatory elements within the rat IgH 3' enhancer, deletion analysis was performed. Transfection experiments using reporter constructs suggest that the enhancer contains three functionally distinct domains, two of which are lymphoid specific and one domain is active in both lymphoid and in nonlymphoid cells. The three domains together contribute to enhancer function and act synergistically. Further analyses suggest that a putative mu E1 site, octanucleotide motif, mu E3 site, and mu B/Ets-like motif are important for the overall transcriptional activity of the IgH 3' enhancer. Moreover, we provide evidence that an additional Ets-like element, micro A, is involved in the tissue specific regulation of enhancer activity and that binding of a protein to this element correlates with the transcriptional activity of one of the lymphoid restricted domains.


An abasic site analogue activates a c-Ha-ras gene by a point mutation at modified and adjacent positions. Synthetic c-Ha-ras genes with an analogue of an abasic site in the first or the second position of codon 12, or in the second position of codon 61 were constructed and transfected into NIH3T3 cells. The genes with the lesions in codon 12 exhibited more focus formation than a normal c-Ha-ras gene, while the gene with the lesion in codon 61 did not. Transformed cells were isolated from the foci, and the c-Ha-ras genes present in the transformants were analysed. A point mutation to A in the modified position was found most frequently in the cases of ras genes modified in codon 12. Surprisingly, point mutations in the adjacent position were also detected. These results indicate that dTMP, and not dAMP, was mainly incorporated into the sites opposite to the abasic site analogue, and that incorrect deoxynucleotides were incorporated in the position adjacent to the abasic site analogue.


Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure. We have recently reported the cloning and sequencing of the gene for the mitochondrial release factor mRF-1. mRF-1 displays high sequence similarity to the bacterial release factors RF-1 and RF-2. A database search for proteins resembling these three factors revealed high similarities to two amino acid sequences deduced from unassigned genomic reading frames in Escherichia coli and Bacillus subtilis. The amino acid sequence derived from the Bacillus reading frame is 47% identical to E.coli and Salmonella typhimurium RF-2, strongly suggesting that it represents B.subtilis RF-2. Our comparison suggests that the expression of the B.subtilis gene is, like that of the E.coli and S. typhimurium RF-2 genes, autoregulated by a stop codon dependent +1 frameshift. A comparison of prokaryotic and mitochondrial release factor sequences, including the putative B.subtilis RF-2, leads us to propose a five-domain model for release factor structure. Possible functions of the various domains are discussed.


Interaction of the erythroid transcription factor cGATA-1 with a critical auto-regulatory element. We have performed a mutational analysis of the promoter for the chicken erythroid-specific GATA-1 transcription factor, and have investigated in detail the interaction of the factor with an upstream auto-regulatory element (ARE). We find that a single proximal GATA binding site of the ARE is required for promoter activity in primary erythroid cells; however, this minimal promoter is inappropriately active in fibroblasts. At least two molecules of GATA-1 can interact with the ARE, and sequences outside of the consensus site appear critical for the transcriptional activity of the bound protein. Finally, we provide evidence for complex protein/DNA interactions at the ARE, including the ability of GATA-1 to bend DNA.


Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells. The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.


Gene dosage as a possible major determinant for equal expression levels of genes encoding RNA polymerase subunits in the hypotrichous ciliate Euplotes octocarinatus. Ciliated protozoa harbor two different types of nuclei in each cell. The diploid micronucleus is the transcriptionally inactive generative nucleus, while the macronuclous contains a highly amplified transcriptionally active genome of lower complexity. The macronuclear genes encoding the two largest subunits of both RNA polymerases I and II of Euplotes octocarinatus were identified by a novel method of two step PCR walking, employing primer pairs derived from telomeric sequences of the organism and known conserved RNA polymerase polypeptide sequences, respectively. The relative gene dosage was determined. The genes are present in equal copy numbers for the respective matching subunits. Northern hybridizations showed comparable amounts of transcripts, as well, within the matching pairs. Mapping of the 5'-termini of the transcripts of the gene sized chromosomes showed that the upstream nontranscribed regions are very short and contain characteristic sequence motifs which could be the determinants of equal promoter strengths for subunits of a common RNA polymerase.


Stoichiometry of the Cre recombinase bound to the lox recombining site. The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.


The mechanisms controlling ribosomal protein L1 pre-mRNA splicing are maintained in evolution and rely on conserved intron sequences. Sequences corresponding to the third intron of the X.laevis L1 ribosomal protein gene were isolated from the second copy of the X.laevis gene and from the single copy of X.tropicalis. Sequence comparison revealed that the three introns share an unusual sequence conservation which spans a region of 110 nucleotides. In addition, they have the same suboptimal 5' splice sites. The three introns show similar features upon oocyte microinjection: they have very low splicing efficiency and undergo the same site specific cleavages which lead to the accumulation of truncated molecules. Computer analysis and RNAse digestions have allowed to assign to the conserved region a specific secondary structure. Mutational analysis has shown that this structure is important for conferring the cleavage phenotype to these three introns. Competition experiments show that the cleavage phenotype can be prevented by coinjection of excess amounts of homologous sequences.


Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing. Functional domains within the mammalian U2 snRNP particle that are required for pre-mRNA splicing have been analysed using antisense oligonucleotides. A comparison of the melting temperatures of duplexes formed between RNA and different types of antisense oligonucleotides has demonstrated that the most stable hybrids are formed with probes made of 2'-O-allyl RNA incorporating the modified base 2-aminoadenine. We have therefore used these 2'-O-allyl probes to target sequences within the central domain of U2 snRNA. Overlapping biotinylated 2'-O-allyloligoribonucleotides complementary to the stem loop Ila region of U2 snRNA (nucleotides 54-72) specifically affinity selected U2 snRNA from HeLa nuclear extracts. These probes inhibited mRNA production in an in vitro splicing assay and caused a concomitant accumulation of splicing intermediates. Little or no inhibition of spliceosome assembly and 5' splice site cleavage was observed for all pre-mRNAs tested, indicating that the oligonucleotides were specifically inhibiting exon ligation. This effect was most striking with a 2'-O-allyloligoribonucleotide complementary to U2 snRNA nucleotides 57-68. These results provide evidence for a functional requirement for U2 snRNP in the splicing mechanism occurring after spliceosome assembly.


Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila. Chromatin footprinting in Drosophila tissue culture cells has detected the binding of a non-histone protein at +8 of the distal Adh RNA start site, on a 10-bp direct repeat motif abutting a nucleosome positioned over the inactive Adh distal promoter. Alternatively the active promoter is bound by a transcription initiation complex. We have characterized and purified a protein Adf-2 that binds specifically to this direct repeat motif 5'TCTCAGTGCA3', present at +8 and -202 of the distal RNA start site. DNase I footprinting, methylation interference, and UV-crosslinking analyses showed that both direct repeats interact in vitro with a nuclear protein of approximately 120 kilodaltons (kDa). We purified Adf-2 through multiple rounds of sequence-specific DNA affinity chromatography. Southwestern analysis showed that the purified 120 KDa polypeptide binds the Adf-2 motif efficiently as a monomer or homomultimer. In vivo titrations of Adf-2 activity with the Adf-2 motif by transient co-transfection competitions in different Drosophila cell lines suggested that Adf-2 is a cell-specific repressor. Adf-2 has been detected ubiquitously in vitro, but is functional in vivo as a sequence-specific DNA binding protein and repressor only in the cells that have the inactive distal promoter. We discuss the possibility that an activation process is required for Adf-2 protein to bind DNA and function in vivo.


Extensive turnover of telomeric DNA at a Plasmodium berghei chromosomal extremity marked by a rare recombinational event. The dynamics of telomere turnover were studied in Plasmodium, whose telomeric structures consist of linear, recognisable sequences of two distinct repeats (TTTAGGG and TTCAGGG). Independent recombinant clones containing a well-defined chromosomal extremity of Plasmodium berghei, both before and after a rare insertion event took place, were obtained from clonal parasite populations and analysed. The insertion, which splits the original telomere and causes a significant reduction in the size of the telomeric structure, is shown to consist of an integer number of subtelomeric repeats typical of P.berghei, flanked on both sides by telomere-derived motifs. Analysis of the telomeric repeat sequence heterogeneity in the otherwise homogeneous populations examined, is compatible with a model in which diversification of a given telomere is driven by the occurrence of breakpoints whose frequency rapidly increases along the telomeric tract when moving in the outward direction. The breakpoints might be due either to terminal deletions followed by random serial addition of the two repeat versions, or to recombination events. The shortening/elongation mechanism is favoured against the recombination hypothesis because of the absence of higher-order patterns in the sequence of telomeric repeats.


Ligand dependence of estrogen receptor induced changes in chromatin structure. To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure. Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol. These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure. These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo.


Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII. A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.


Spontaneous chromosome rearrangements in the protozoan Giardia lamblia: estimation of mutation rates. Subcloned lines of the WB strain of Giardia lamblia contain polymorphic ribosomal RNA (rRNA) encoding chromosomes (Le Blancq et al., Nucl. Acids Res. 1991, 19, 4405-4412). We show that in a continuously propagated culture of G.lamblia trophozoites the proportion of trophozoites with rearranged rRNA encoding chromosomes gradually increases, consistent with the high mutation rate of about 1% per cell per division cycle. This conclusion is based on the finding in one experiment that after about 8 division cycles 20% of the population consisted of independent mutants, while after approximately 100 division cycles 87.5% of the population were independent mutants. In a second experiment, approximately 38% and 71.5% of the trophozoites were independent mutants after approximately 9 and approximately 100 division cycles, respectively. The data show that the genome of the WB strain of G.lamblia has a highly recombinogenic phenotype. Extensive karyotype heterogeneity has also been observed among recently isolated G.lamblia strains obtained from a defined geographic area (Korman et al., J. Clin. Invest. 1992, 89, 1725-1733) suggesting that a high mutation rate might also occur in vivo.


Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide. An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods. Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry. After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination. The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer. E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer. E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.


Gilvocarcin V exhibits both equilibrium DNA binding and UV light induced DNA adduct formation which is sequence context dependent. The relative degree of both equilibrium binding and of ultraviolet light induced adduct formation for the antitumor antibiotic gilvocarin V with two hexaecamer DNA sequence isomers, d[ATATATAGCTATATAT]2 and d[AAAAAAAGCTTTTTTT]2, was assessed. The experiments reveal that gilvocarin V binds, under equilibrium conditions, and reacts, in the presence of exogenously applied UV light, more efficiently with the alternating purine:pyrimidine sequence hexadecamer than the homopurine:homopyrimidine duplex at identical gilvocarcin V to DNA duplex ratios. DNAse I digests of adduct containing duplexes derived from the d[AAAAAAAGCTTTTTTT]2 duplex, identified and isolated using gel shift assays employing denaturing polyacrylamide gels, confirm that gilvocarcin V adducts can be formed with thymine residues but suggest that adduct formation with either adenine or guanine residues is also possible.


Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo. Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.


Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.


Construction of human XRCC1 minigenes that fully correct the CHO DNA repair mutant EM9. The human gene that corrects the DNA repair defect of the CHO cell mutant EM9 is designated XRCC1 and is the first human gene to be cloned that has an established role in DNA strand-break repair. In this study, either an XRCC1 cosmid genomic fragment or synthetic oligonucleotides were ligated to an incomplete XRCC1 cDNA to generate two full-length XRCC1 cDNA constructs. The ability of these minigene constructs to restore normal levels of sister chromatid exchange (SCE) to EM9 upon transfection was demonstrated, and the transfectants grew at normal rates in selective medium that is fully toxic to EM9 cells. Constructs in which the XRCC1 open reading frame (ORF) was transcribed from the SV40 early promoter or the genomic XRCC1 native promoter were compared in their efficiency of correction. EM9 transfectants derived from the SV40 promoter displayed fewer SCEs and lower sensitivity to CldUrd than either AA8 wild-type cells or transfectants containing the ORF transcribed from the native promoter.


Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication--potential effectiveness against most presently sequenced HIV-1 isolates. Several mono-, di-, tetra-, penta- and nonaribozymes were developed. These multitarget-ribozymes were targeted to cleave HIV-1 env RNA at up to nine different conserved sites. Each multitarget-ribozyme consisted of a chain of up to nine hammerhead motifs, each flanked by a different targeting sequence. The multitarget-ribozymes were functional in vitro and gave rise to multiple, specific partial and/or complete RNA digestion products. Per RNA copy, multitarget-ribozymes were more efficient than monoribozymes or ribozymes targeting a subset of the same sites. In contrast to monoribozymes, a 400nt nonaribozyme, targeted to cleave at nine different sites within a 1.3kb HIV-1 env RNA substrate, was active and showed the same specificity of cleavage when it was part of a large 3.3kb transcript. We conclude that multitarget-ribozymes retain the specificity of monoribozymes, but they are more efficient per ribozyme RNA copy and they remain active when they are part of a large transcript. A tetra-, penta- or nonaribozyme under control of the SV40 late promoter, the beta-actin gene promoter or the HIV-1 LTR, respectively, were cotransfected with the infectious HIV-1 DNA clone pNL4-3 into permissive HeLa T4 cells. Each cotransfection resulted in a specific inhibition of HIV-1 replication as determined by syncytia formation and p24 antigen release. In addition, coexpression of the nonaribozyme with an HIV-1 env RNA transcript resulted in the specific dramatic reduction of the env transcript. We conclude that the multitarget-ribozymes are also functional intracellularly. A nucleotide sequence comparison of the target sites indicates that the multitarget-ribozymes could potentially be effective against all thirty HIV-1 isolates presently sequenced. Their use may help to slow the selection of viral escape mutants and thereby prolong their effectiveness. We anticipate that multitarget-ribozymes will also be more effective in the successful targeting of less variable cellular RNAs.


Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences. A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.


Integration of image analysis and robotics into a fully automated colony picking and plate handling system. We describe here the integration of image analysis and robotics to produce a fully automated colony picking/plate handling system. Biological tests were performed to verify its performance in terms of sterilisation and accuracy of picking. The machine was then used by a single operative to pick a 36,000 clone cDNA library in approximately 42 hrs over 5 days.


Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Transcription factors containing the POU-domain have been shown to be important regulators of tissue-specific gene expression in the pituitary and lymphoid cells. Using a polymerase chain reaction (PCR)-based strategy, we have searched for similar factors that may be expressed in adult human pancreatic islets. This approach resulted in the amplification of sequences encoding the octamer binding proteins Oct1 and Oct3 (also called Oct4). The isolation of cDNAs encoding Oct3 revealed the expression of two isoforms of this transcription factor termed Oct3A and Oct3B that are generated by alternative splicing. Human Oct3A and Oct3B are composed of 360 and 265 amino acids, respectively, of which the 225 amino acids at the COOH-termini are identical. The sequence of human Oct3A shows 87% amino acid identity with mouse Oct3. Reverse-transcriptase PCR showed low levels of expression of both Oct3A and Oct3B mRNA in all adult human tissues examined. We also isolated and characterized the human Oct3 gene (OTF3) and a related gene, OTF3C. The human Oct3 gene, localized to human chromosome 6 in the region of the MHC complex, spans about 7 kb and consists of five exons. The Oct3-related gene, OTF3C, is a retroposon and has been localized to human chromosome 8. Southern blotting and PCR amplification of human DNA indicated the presence of other OTF3-related genes as has been previously noted in the mouse. Two polymorphisms which can be typed using PCR were identified in OTF3 which will facilitate genetic studies of this gene.


Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates. Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides.


Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. The leader (called omega) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes. Although little secondary structure is predicted to exist within omega, the primary sequence of the 68 base leader is highly organized. Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of omega. In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within omega. We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement. In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs. beta-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation. A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical. Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing. Thus, these two motifs are functionally redundant.


Cloning of human and bovine homologs of SNF2/SWI2: a global activator of transcription in yeast S. cerevisiae. We performed positional cloning of genes carried on yeast artificial chromosomes that span a human translocation breakpoint associated with a human disease and isolated by chance human and bovine genes with strong homology to the S. cerevisiae genes, SNF2/SWI2 and STH1, and the D. melanogaster gene brahma. We report here sequence analysis, expression data, and functional studies for this human SNF2-like gene (hSNF2L) and its bovine homolog (bovSNF2L). Despite strong homology at the amino acid level, hSNF2L is not capable of complementing the yeast mutations snf2 or sth1 in S. cerevisiae. Furthermore, in contrast to SNF2 itself, a fusion protein consisting of the DNA binding domain of LexA and hSNF2L did not transactivate a reporter gene downstream of LexA binding sites in a yeast expression system. The strong similarity between hSNF2L and these yeast and drosophila genes suggest that the mammalian genes are part of an evolutionarily conserved family that has been implicated as global activators of transcription in yeast and fruitflies but whose function in mammals remains unknown.


A novel strategy for site-directed chemical reactions in single stranded DNA--absorption and NMR spectroscopic studies of model compounds. A new and simple model enabling a chemical species to be brought to a preselected site in single strand DNA is reported. Two oligonucleotides containing a propanediol linkage were hybridized to their complementary sequences with an extra-base opposite the propanediol derivative. Absorption studies results shown that the addition of a bisacridine derivative strongly increased the stabilities of both duplexes when added in a 1:1 ratio. NMR studies on one of these duplexes brought evidence of the intercalation of the bisacridine at the position involving the propanediol linkage. These results suggest that this system could be used to target a specific reaction at a preselected position using the bisacridine derivative as carrier for the reactive species.


Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis. In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.


A new approach to the analysis of DNase I footprinting data and its application to the TFIIIA/5S DNA complex. We have re-examined DNase I footprinting data for the binding of transcription factor IIIA (TFIIIA) to the 5S RNA gene, taking into account the protein-DNA contacts observed in the crystal structure of the DNase I/DNA complex (1, 2). This structure was not available when many of the original footprinting experiments on the TFIIIA/DNA complex were performed. In this way the pattern of DNase I cleavage can be interpreted to map out with greater precision the regions on the 5S DNA occupied by TFIIIA. Then, assuming the binding site for a zinc-finger may be the same as that found in the structure of the zinc-finger protein Zif268/DNA complex (3), and taking into account footprinting data for truncated forms of TFIIIA, the TFIIIA zinc-fingers were fitted within the permitted regions. On the basis of this, an alignment of the zinc-fingers of TFIIIA with its DNA binding site is proposed, which combines features of earlier models (4).


The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference. We show that the single intron of the actin gene of the yeast Saccharomyces cerevisiae contains a cryptic promoter for transcription of the second exon. This promoter is inactive in the normal actin gene, but can be activated when the actin gene promoter is deleted. An identical activation was induced by placing efficient transcriptional terminators at position 61 of the 309 bp intron. In all cases transcripts with identical 5' ends close to the boundary of the intron and the second exon were produced. These results indicate that the cryptic promoter in the actin intron is occluded in the normal actin gene by transcriptional interference with the actin gene promoter. Transcription initiation near the intron/exon 2 boundary is enabled by protection from traversing polymerases, that initiated transcription at the upstream located actin gene promoter. A partial promoter protection using leaky terminators resulted in small amounts of transcripts initiated from the cryptic promoter. Although we do not know any function of the cryptic promoter in actin gene expression, it is tentative to speculate that the cryptic intron promoter might be a relict of a promoter that was functional earlier in evolution.


Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases. The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).


Heteronuclear NMR of DNA with the heteronucleus in natural abundance: facilitated assignment and extraction of coupling constants. Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.


The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer. A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.


Cooperative effects of C/EBP-like and NF kappa B-like binding sites on rat serum amyloid A1 gene expression in liver cells. Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to inflammation, its expression is increased by 1000-fold, primarily because of a 200-fold increase in the rates of SAA gene transcription. We have shown that when 304 bp of 5' flanking region of the rat SAA1 gene is fused to a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, CAT activity is induced in a cell-specific manner in response to conditioned media prepared from activated mixed lymphocyte cultures and recombinant interleukin-1. In this study, deletion of the SAA1 promoter to -120 bp with respect to the transcriptional start site did not diminish promoter activity; however, deletion to -94 bp renders the promoter completely inactive. Functional analysis have demonstrated that a 66-bp DNA fragment spanning -138 bp to -73 bp could confer cytokine responsiveness to a heterologous thymidine kinase promoter. Within this 66-bp responsive element resided an NF kappa B-like-binding site and a C/EBP-like-binding site. Although each binding site alone could confer responsiveness when stimulated with conditioned media and TPA, the response was much weaker than that observed when both sites were present. Moreover, site-specific mutations of either binding site completely abolished SAA1 promoter activity. Taken together, these results suggest a functional importance for and cooperative interaction of these two nuclear-factor binding sites in the cytokine-induced expression of the rat SAA1 gene.


Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control. Expression of the rpoBC genes encoding the beta and beta' RNA polymerase subunits of Escherichia coli is autogenously regulated. Although previous studies have demonstrated a post-transcriptional feedback mechanism, complex transcriptional controls of rpoBC expression may also contribute. We show that an attenuator (rpoBa) separating the ribosomal protein (rpl) genes from the rpoBC genes in the rplKAJLrpoBC gene cluster is modulated in its efficiency in response to changes in the frequency of transcription initiated by promoters located upstream. A series of rplJLrpoBalacZ transcriptional fusions was constructed on lambda vectors in which transcription into the rpoBa attenuator was varied by using a variety of promoters with different strengths. beta-galactosidase assays performed on monolysogens of the recombinant phage show that with transcription increasing over a 40-fold range, readthrough of rpoBa decreases from 61% to 19%. In contrast, two other well-characterized terminators show nearly constant efficiencies over a similar range of transcription frequencies. Using a set of phage P22 ant promoter variants with single-nucleotide changes in the promoter consensus sequences also demonstrates that the modulation of rpoBa function appears to be unrelated to the phenomenon of 'factor-independent antitermination' reported by others. The implications for autogenous control of RNA polymerase synthesis are discussed.


Characterisation of a boundary between satellite III and alphoid sequences on human chromosome 10. Alphoid and satellite III sequences are arranged as large tandem arrays in the centromeric regions of human chromosomes. Several recent studies using in situ hybridisation to investigate the relative positions of these sequences have shown that they occupy adjacent but non-overlapping domains in metaphase chromosomes. We have analysed the DNA sequence at the junction between alphoid and satellite III sequences in a cosmid previously mapped to chromosome 10. The alphoid sequence consists of tandemly arranged dimers which are distinct from the known chromosome 10-specific alphoid family. Polymerase chain reaction experiments confirm the integrity of the sequence data. These results, together with pulsed field gel electrophoresis data place the boundary between alphoid and satellite III sequences in the mapping interval 10 centromere-10q11.2. The sequence data shows that these repetitive sequences are separated by a partial L1 interspersed repeat sequence less than 500bp in length. The arrangement of the junction suggests that a recombination event has brought these sequences into close proximity.


Repair of UV-induced (6-4)photoproducts measured in individual genes in the Drosophila embryonic Kc cell line. The nucleotide excision repair (NER; dark-repair) of (6-4)photoproducts ((6-4)PPs) was assayed in cells from a permanent Drosophila melanogaster embryonic cell line, Kc, after exposure to 20 or 40 J/m2 ultraviolet (UV) light. Induction rates in the transcriptionally active genes Gart and Notch as well as in the inactive white locus is similar. They are formed with a frequency of about one-third of that of cyclobutane pyrimidine dimers (CPDs). In all three genes, (6-4)PPs are repaired with the same rate and to the same extent: 31% of the (6-4)PPs are removed in 4 hours post-irradiation and after 16 hours repair is nearly complete. In none of the three genes strand-specific repair was found. Exposure of cells that were irradiated with 40 J/m2 UV to photoreactivating light for 1 hour prior to dark-repair incubation, resulted in enhanced repair of (6-4)PPs.


End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells. We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.


Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta. The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.


How M.MspI and M.HpaII decide which base to methylate. The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.


Isolation of genomic DNA encoding transcription factor TFIID from Acanthamoeba castellanii: characterization of the promoter. We have isolated a genomic clone encoding Acanthamoeba castellanii TFIID. The clone contains the entire TFIID gene, 300 bp of 5' promoter sequences and several hundred base pairs of 3' non-coding sequence. The coding region is interrupted by two short introns, but is otherwise identical to Acanthamoeba TFIID cDNA. Comparisons between forty four Acanthamoeba intron 5' and 3' boundaries suggest a 5' splice site consensus of GTACG(T/C) and a 3' consensus of CAG. We determined the position of the transcription initiation site used in vivo, and show that the same site is used in vitro by homologous nuclear extracts. Deletion analysis of the promoter region shows that the minimal promoter required for efficient expression in vitro is located between -97 and +4 relative to the transcription start site. Three regions within the promoter are important for transcription in vitro; sequences between -97 and -35, the TATAAA box and the initiation region. The initiation region is dispensable but appears to position the transcription start site relative to the TATAAA box. The TATAAA box is absolutely required for transcription initiation whereas the upstream region stimulates transcription approximately five-fold.


Lability of monofunctional cis-platinum adducts: role of DNA double helix. Recently, we have shown that the adduct formed in the reaction between the platinum-triamine complex cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl]2+ and one single-stranded oligonucleotide was stable but became labile as soon as the platinated oligonucleotide was paired with its complementary strand (Gaucheron et al. Proc. Natl. Acad. Sci. USA 88, 3516-3519 (1991)). To generalize this finding we have now studied large DNA fragments containing several adducts. The stability of the adducts within single-stranded DNA is demonstrated by absorption spectrophotometry and by replication mapping experiments. Several approaches are used to prove the lability of the adducts within double-stranded DNA. Replication mapping experiments reveal that an unmodified single-stranded DNA when mixed with double-stranded DNA modified by the platinum-triamine complex behaves as a single-stranded DNA modified by the triamine complex. After double-stranded DNA is modified by the platinum-triamine complex, intrastrand and interstrand cross-links are progressively formed during subsequent incubation as revealed by transcription mapping experiments and gel electrophoresis under denaturing conditions. Finally, replication mapping experiments show that the lability of the adducts within a double-stranded DNA depends upon the nature of the flanking nucleotide residues. All these results support the proposal that the DNA double helix acts as a catalyst in the reaction between DNA, cis-diamminedichloroplatinum(II) and N-methyl-2,7-diazapyrenium.


Detection of single base differences using biotinylated nucleotides with very long linker arms. A simple primer extension method for detecting nucleotide differences is based on the substitution of mobility-shifting analogs for natural nucleotides (1). This technique can detect any single-base difference that might occur including previously unknown mutations or polymorphisms. Two technical limitations of the original procedure have now been addressed. First, switching to Thermococcus litoralis DNA polymerase has eliminated variability believed to be due to the addition of an extra, non-templated base to the 3' end of DNA by Taq DNA polymerase. Second, with the analogs used in the original study, the mobility shift induced by a single base change can usually be resolved only in DNA segments 200 nt or smaller. This size limitation has been overcome by synthesizing biotinylated nucleotides with extraordinarily long linker arms (36 atom backbone). Using these new analogs and conventional sequencing gels (0.4 mm thick), mutations in the human beta-hexosaminidase alpha and CYP2D6 genes have been detected in DNA segments up to 300 nt in length. By using very thin (0.15 mm) gels, single-base polymorphisms in the human APOE gene have been detected in 500-nt segments.


Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA. 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA.


The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: conservation and variations among vertebrates. The complete mitochondrial (mt) genome of Crossostoma lacustre, a freshwater loach from mountain stream of Taiwan, has been cloned and sequenced. This fish mt genome, consisting of 16558 base-pairs, encodes genes for 13 proteins, two rRNAs, and 22 tRNAs, in addition to a regulatory sequence for replication and transcription (D-loop), is similar to those of the other vertebrates in both the order and orientation of these genes. The protein-coding and ribosomal RNA genes are highly homologous both in size and composition, to their counterparts in mammals, birds, amphibians, and invertebrates, and using essentially the same set of codons, including both the initiation and termination signals, and the tRNAs. Differences do exist, however, in the lengths and sequences of the D-loop regions, and in space between genes, which account for the variations in total lengths of the genomes. Our observations provide evidence for the first time for the conservation of genetic information in the fish mitochondrial genome, especially among the vertebrates.


Two regions in human DNA polymerase beta mRNA suppress translation in Escherichia coli. Although human DNA polymerase beta (DNA pol beta) shows 96% identity with rat DNA pol beta at the amino acid level, it is weakly expressed in Escherichia (E.) coli relative to the rat enzyme. The mechanism of this suppression was investigated. Pulse-chase protein labeling and steady state mRNA analysis showed that mature human DNA pol beta protein is relatively stable in E. coli and the levels of human and rat DNA pol beta mRNA were comparable indicating that the human DNA pol beta expression is suppressed at the translational level. By systematic expression analysis of a number of chimeric genes composed of human and rat cDNAs, two strong translational suppression regions were mapped in the human DNA pol beta mRNA; one was named TSR-1, corresponding to CGG encoding arginine (arg) at position 4 and the other, termed TSR-2, is located between codons 153 and 199. Since substitution of the rat Arg-4 codon with synonymous codons showed strong effects upon the expression level, we propose that the arg codon at the N-terminal coding region plays a role in modulating expression.


Activation and repression sequences determine the lens-specific expression of the rat gamma D-crystallin gene. Rat lens nuclear extracts contain a factor that binds to position -57 to -46 of the rat gamma D-crystallin promoter region. This factor protects the sequence 5'-CTGCCAACGCAG-3' in a footprint analysis. Binding to this region is crucial for maximal promoter activity in rat lens cells, but this sequence was unable to act as an enhancer when cloned in front of a heterologous promoter. A region directly upstream from this activating sequence, between position -85 to -67, acts as a strong silencer of promoter activity in non-lens cells. This silencing effect is mediated by trans-acting factor(s). Our data provide evidence for two regulatory elements in rat gamma D-crystallin gene expression, an activating sequence active in lens cells and a silencing sequence active only in non-lens cells. The factor that binds to the activating sequence could be detected only in lens cells and may be a determinant of the lens-specific expression of the gamma-crystallin genes.


Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples. We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.


Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA. We have studied the effect of intermolecular triplexes formation on the yield of cyclobutane photodimers in DNA. DNA duplex within the pyrimidine-purine-pyrimidine triplex d(TC)nd(GA)nd(CT)n is protected from the formation of cyclobutane photodimers in the case of the stabilization of this triplex by acid pH, and in the case of supplementary stabilization by Mg2+ or Zn2+. We have studied pH-independent pyrimidine-purine-purine triplexes stabilized by bivalent cations. In such triplexes, the protection from the formation of [6-4] photodimers is observed, whereas the protection from cyclobutane dimer formation does not take place. The formation of the d(TC)nd(GA)nd(GA)n triplex leads to an inversion of the intensities of cyclobutane CT and TC photodimers. We observed a sharp decrease in photoreactivity with respect to cyclobutane dimers in the duplex tract d(C)18d(G)18 in the presence of Ba2+, Cd2+, Co2+, Mn2+, Zn2+ and Ni2+. The formation of the d(C)nd(G)nd(G)n triplex leads to 'antifootprinting', i.e. an increase in the yield of cyclobutane photodimers.


The transcriptional start site for a human U6 small nuclear RNA gene is dictated by a compound promoter element consisting of the PSE and the TATA box. Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes, but their respective roles in initiation site selection are unknown. We have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription. The spacing requirement between the two elements is highly stringent, implying a possible interaction between the factors that bind them. Our results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, we suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the human U6 gene promoter.


The 66 kDa component of yeast SFI, stimulatory factor I, is hsp60. DNA polymerase epsilon stimulatory factor I (SFI) has been shown to contain three peptides, p66, p37 and p13. Two of these components have been identified. The p66 gene was cloned by using a p66 antibody to screen a lambda gt11 library. A portion of the gene was sequenced and confirmed to encode p66 by the presence of protein sequence corresponding to that of p66 tryptic peptides. The gene was identified as HSP60 by a homology search of GenBank. Tryptic peptides of p37 were sequenced and identified as belonging to yeast translation initiation factor 4A by a homology search of PIR. The HSP60 gene maps to chromosome XII.


Alw26I, Eco31I and Esp3I--type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA. The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.


Nutritional regulation of nucleosomal structure at the chicken malic enzyme promoter in liver. Transcription of the chicken malic enzyme gene in the liver is stimulated by feeding and inhibited by starvation. Concomitant with the increase in transcription caused by refeeding, chromatin structure around the transcription start site of the malic enzyme gene is modified in the liver. Digestion of chromatin in isolated nuclei with DNase I revealed four feeding-induced DNase I hypersensitive sites (-220, -170, -130 and -70 bp) near the malic enzyme promoter. Similarly, digestion of chromatin with restriction endonucleases detected enhanced cleavage within this region when birds were refed. Micrococcal nuclease detected the presence of nucleosomes over this region in the starved state, but not in the fed state. After food was withdrawn from fed birds, nucleosomes were reformed in this region within 6 h. The speed and magnitude of the changes in nucleosomal structure in this region suggest that they did not require DNA replication.


Site-specific cleavage of IGF-II mRNAs requires sequence elements from two distinct regions of the IGF-II gene. The human insulin-like growth factor II (IGF-II) gene constitutes a complex transcriptional unit that contains nine exons and four promoters. Expression of the IGF-II gene yields a family of mRNAs that all encode prepro-IGF-II. In addition, a stable 1.8 kb RNA is formed that is derived from the 3' untranslated region of exon 9. Recently, we have shown that this RNA species arises by site-specific endonucleolytic cleavage of IGF-II mRNAs and not by transcription from a separate promoter. In the present study we establish that two widely separated sequence elements of approximately 300 nucleotides, both located within exon 9, are required for this cleavage reaction. The first element encompasses about 200 nucleotides upstream and 100 nucleotides downstream of the cleavage site, while the second element is located within a region of 330 nucleotides about 2 kb upstream of the cleavage site. Interestingly, site-specific cleavage also occurred when a fragment from exon 9 of the IGF-II gene containing these two elements was inserted into the 3' untranslated part of the beta-globin gene. Apparently, the expressed hybrid beta-globin-IGF-II mRNA contains all the regulatory elements to confer site-specific endonucleolytic cleavage.


Specificities of three tight-binding Lac repressors. Tight binding mutants of Lac repressor exhibit complex repression phenomena. In this work, in vivo Lac operator binding of three such mutants of E. coli Lac repressor (X86: ser 61-leu, l12: pro 3-tyr and the double mutant l12X86: pro 3-tyr, ser 61-leu) was analyzed. Repression of beta-galactosidase synthesis controlled by ideal lac operator and its 27 symmetric operator variants containing each possible base-pair at each single half-operator position in the presence of the tight-binding Lac repressor mutants was determined. The average increase of repression with all operator variants was about 3 fold with the X86 mutant. It was about 4 fold with the l12 mutant and about 2 fold with the double mutant l12X86 as compared to wildtype Lac repressor. The X86 mutant showed the same increase of affinity to all operator variants, whereas the l12 and l12X86 mutants exhibited lower repression with some variants than with most others. These results suggest that the X86 mutant has gained no additional specificity. In contrast the l12 mutant and the l12X86 mutant exhibit a relaxed specificity for certain base pairs in positions 1 and 3 of lac operator. This suggests that the extreme N-terminus of Lac repressor may interact with the inner base-pairs in the minor groove.


Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene. Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively. Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains. The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis. The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da). The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases. Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da). This ORF is suggested to encode the corresponding endonuclease R.MthZI.


Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA. Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle.


A rapid chemiluminescent method for quantitation of human DNA. A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.


Hydrolysis of oligoribonucleotides: influence of sequence and length. The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis.


Nonenzymatic hydrolysis of oligoribonucleotides. Selective cleavage of phosphodiester bonds in RNA is important in the processing of large RNA molecules. This paper reports specific cleavage at UA sequences in single stranded oligoribonucleotides as short as hexamers. The hydrolysis between U and A leaves a 2',3'-cyclic phosphate on the 5'-side and a 5'-hydroxyl group on the 3' side of the cleavage. The hydrolysis is promoted by a wide range of cofactors, including polymeric organic compounds such as polyvinylpyrrolydone (PVP) and by proteins. A variety of experiments suggests the cleavage is not due to contamination by ribonuclease. The rate of cleavage is a function of oligoribonucleotide, PVP and spermidine concentrations. Mg2+ is not required. The phenomenon described here can potentially provide a relatively simple way of coding chemical stability into single stranded RNA based on its sequence and structure. This process seems to be similar to that involved in post-transcriptional degradation of mRNA.


Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. Methylated DNA in vertebrates is associated with transcriptional repression and inactive chromatin. Two activities have been identified, MeCP1 and MeCP2, which bind specifically to DNA containing methyl-CpG pairs. In this report we characterize MeCP2. We show that it is more abundant than MeCP1, is more tightly bound in the nucleus, and is distinguishable chromatographically. The two proteins share widespread expression in somatic mammalian cells, and barely detectable expression in early embryonic cells. DNAs containing thymidine which has a methyl group at position 5 are not ligands for the MeCPs. The possible role of MeCP2 in methylation-associated gene inactivation was tested in in vitro transcription extracts. Purified MeCP2 inhibited transcription from both methylated and nonmethylated DNA templates in vitro, probably due to the presence of nonspecific DNA binding domains within the protein. We hypothesise that MeCP2 normally binds methylated DNA in the context of chromatin, contributing to the long-term repression and nuclease-resistance of methyl-CpGs.


Clathrin light chain B: gene structure and neuron-specific splicing. The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing.


Modified DNA fragments activate NaeI cleavage of refractory DNA sites. Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.