Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis.

Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 bp fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1-5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS: a heterodimeric beta-ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a beta-ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORF1-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.


Homologous genes for the C4 isoform of phosphoenolpyruvate carboxylase in a C3 and a C4 Flaveria species.

The C4 isoform of phosphoenolpyruvate carboxylase (PEPCase) in Flaveria trinervia is encoded by the ppcA subgroup of the PEPCase gene family and is abundantly expressed in the mesophyll cells of leaves. The homologous ppcA genes in the C3 plant F pringlei are only weakly expressed and their transcripts do not show the strictly leaf-specific accumulation pattern observed for the F. trinervia genes. Two representative members of the ppcA subfamilies of F. trinervia (C4) and F. pringeli (C3)-named ppcA1-were characterized by Southern blotting, nucleotide sequencing and primer extension analysis. Comparison of the deduced amino acid sequences reveals a close similarity between C4 and C3 isoforms. Only few C4-specific positions can be detected when all known plant PEPCases are included in the comparison. A regulatory domain involved in light-dependent phosphorylation/dephosphorylation of the C4 and crassulacean acid metabolism (CAM) isoforms is present in the ppcA1 gene products of both the C3 and C4 Flaveria. The 5' flanking regions are essentially homologous. The putative promoter regions share several identical sequence motifs (CCAAT, AT-1 and GT-1 box III/III* elements). Additionally, alterations in elements that could contribute to differences in expression rates and light regulation are found. The significance of these findings is discussed with respect to the molecular evolution of C4 photosynthesis in Flaveria.


Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex.

The primary structure of Bacillus subtilis 105 kDa 2-oxoglutarate dehydrogenase (E10) was deduced from the nucleotide sequence of the odhA gene and confirmed by N-terminal sequence analysis. The protein is highly homologous to E1o of Azotobacter vinelandii and Escherichia coli and of bakers' yeast cells. The 5' end of the odhAB mRNA was determined and the promoter region for the odhAB operon was localized to a 375 bp DNA fragment. The cellular concentration of the 4.5 kb odhAB transcript was found to be growth stage dependent; its concentration during growth in nutrient sporulation medium decreased abruptly at the end of the exponential growth phase and it was not detectable in early stationary phase. This decrease in the cellular concentration of the transcript is not the result of an increased rate of decay of the full-length odhAB mRNA, suggesting that transcription is down-regulated at the end of the exponential growth phase. The cellular concentration of the odhA and odhB gene products, E1o and dihydrolipoamide transsuccinylase (E2o), remains essentially constant throughout the growth curve in nutrient sporulation medium, indicating that both are rather stable proteins. In exponentially growing cells, glucose in nutrient sporulation medium repressed the cellular concentration of the odhAB mRNA, as well as that of E1o and E2o, about four-fold. This effect is most likely the result of a decreased rate of transcription from the odhAB promoter, since neither the stability nor the 5'-end of the transcript were affected by glucose in the medium. It is concluded that the cellular concentration of the 2-oxoglutarate dehydrogenase multienzyme complex (E1o and E2o) is regulated mainly at the transcriptional level.


Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.


Asymmetric somatic cell hybridization in plants. I. The early effects of (sub)lethal doses of UV and gamma radiation on the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgaris L.) protoplasts.

An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700-4200 J/m2 and those of 60Co gamma radiation over the range 0.15-1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m2 UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.


Asymmetric somatic cell hybridization in plants. II. Electrophoretic analysis of radiation-induced DNA damage and repair following the exposure of sugarbeet (Beta vulgaris L.) protoplasts to UV and gamma rays.

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.


Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis.

The ptsG gene of Bacillus subtilis encodes Enzyme IIGlc of the phosphoenolpyruvate: glucose phosphotransferase system. The 3' end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes IIIGlc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5' end. These results, combined with the sequence of the 3' end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes IIGlc and IINag of E. coli but which are present in the IIGlc-like protein encoded by the E. coli malX gene.


Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases.

In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated. The major protease, which is responsible for 80-85% of the total activity, is aspergillopepsin A, a protein of ca. 43 kDa, the activity of which is inhibited by pepstatin. In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin. Several protease-deficient mutants were obtained by in vivo UV mutagenesis. In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy. In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted. In three UV-induced mutants, aspergillopepsin A is also missing. One of these mutants, AB1.18, is mutated in the pepA gene, which is located on chromosome I. One of the other mutants, AB1.13, which has only 1-2% of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B. The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene. Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII. Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.


Use of synthetic peptides for the detection and quantification of autoantibodies.

The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins). Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B' synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity. Synthetic peptide antigens have been used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications.(ABSTRACT TRUNCATED AT 250 WORDS)


[Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex].

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.


[Synthesis of a bovine adrenodoxin precursor in vitro and its import into yeast mitochondria].

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.


[Identification of the gene for the immunodominant p35 protein from vaccinia virus].

A major immunodominant envelope protein p35 of vaccina virus was purified by means of extraction from virions with detergent NP-40. The protein was cleaved with CNBr, four homogenous peptides were isolated and their N-terminal amino acid sequences were determined. Computer search in a protein sequences data bank revealed that the immunodominant protein p35 of vaccinia virus is encoded by H3 gene in HindIII-H fragment of vaccinia virus genome.


[Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells].

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.


[Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions].

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.


[Introduction of statistical mutations into strictly defined areas of the genome using a modified oligonucleotide].

A method for inducing mutations in the short region of a gene is suggested. The method involves oligonucleotide modification by hydroxylamine derivative, in vitro enzymatic synthesis of double-stranded DNA using modified oligonucleotide as a primer and selection of mutant colonies using the starting unmodified oligonucleotide as a probe.


[Study of the structure of human serum albumin, liberated from fatty acids, by a tritium marker method].

To elucidate the natural fatty acids effect on the human serum albumin (HSA) structure a new method of tritium labelling was used. The main peculiarity of the method consists in the possibility to get information on the qualitative and quantitative amino acid composition of the surface layer of the protein globule at different conformational states of the globule. Defatted HSA was shown to be characterized a higher accessibility of Asx, Glx, Thr, Ser, Gly, Pro, Ile, Tyr residues while the other residues remain unchanged. Asx residues are characterized by the largest changes (about 8 folds). Full accessible protein surface during defatting increases from 39,000 to 48,000 A2. Fatty acids connected with albumin in the relation 1-3 moles/mol of protein are noted to be the factor increasing the globule compactness and stipulating for the conformational protein stability to warmth, urine and guanidine salts effect.


[Nucleotide sequence of the pNB2 plasmid from thermophilic bacteria Clostridium thermosaccharolyticum].

The complete nucleotide sequence of pNB2, a 1.9-kilobases cryptic plasmid from thermophilic Clostridium thermosaccharolyticum has been determined. The plasmid consists of 1882 base pairs and has a G+C composition of 27.2%. The sequence contains three open reading frames capable of coding for polypeptides two of which were identified in maxicell Escherichia coli extracts. Our future studies are directed toward a construction of pNB2-derivatives as vectors for Clostridia.


[Inhibition of reproduction of the human immunodeficiency virus in cell culture by 2',3'-dideoxynucleoside-5'-phosphites].

5'-Phosphites (5'-hydrogenphosphonates) of 2',3'-dideoxynucleosides (T, A, G, C) were synthesized and studied as inhibitors of human immunodeficiency virus type 1 (HIV-1) in MT4 and CEM13 cell cultures. It was shown that all 5'-phosphites effectively inhibit the production of viral antigens and protect cells from the cytotoxic effect of HIV infection. 5'-Phosphites were more active antiviral compounds than the corresponding nucleosides.


[Expression of human urokinase in inoculated rodent cells. Loss of introns during gene transfer using a retroviral vector].

Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.


[Analogs of nucleotides, modified by a sugar residue and pyrimidine base, in a DNA synthesis reaction, catalyzed by Thermus aquaticus DNA polymerase].

Substrate properties of dNTP analogues in the DNA synthesis reaction catalyzed by Thermus aquaticus DNA polymerase were studied. It was shown that most of dNTP analogues which were known as terminators of DNA synthesis of E. coli DNA polymerase I were able to terminate DNA synthesis catalyzed by Thermus aquaticus DNA polymerase. An interesting feature of Thermus aquaticus DNA polymerase was the ability to utilize 3'-azido-2',3'-dideoxythymidine triphosphate as terminating substrate. Relative efficiency of tested dNTP analogues incorporation into the DNA growing chain was estimated.


[Single-stranded DNA from viruses, prokaryotes and eukaryotes].

The review presents the results of investigations of single-stranded DNAs of viruses, bacteria and cells of higher organisms. Methods of revealing, isolating and analysis of these DNAs are presented. A large variety of single-stranded DNA containing genomes of plant and animal viruses was revealed. Attention is drawn to the integration and replication of viral genomes. Results of SV40 integration during the first two days after infection of Chinese hamster cells are shown. Results of studying multi-copy single-stranded DNA in bacterial cells were analysed. In separate sections, the replication of plasmid single-stranded DNA was studied as well as the problem of plasmid stability in cells. Advances in bacteria transformation studies are stated. Data of single-stranded DNA investigation in cells of higher organisms are mainly presented on the example of early embryos. Data on the analysis of gene hypersensitivity to nuclease S1 are given. A table of proteins destabilizing and unweaving single-stranded DNA and a classification table of proteins bound with single-stranded DNA according to their functional significance are presented. It is stated that the problem of single-stranded DNA significance in cells remains open, although some results have been achieved.


COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae.

The COT1 gene of Saccharomyces cerevisiae has been isolated as a dosage-dependent suppressor of cobalt toxicity. Overexpression of the COT1 gene confers increased tolerance to cobalt and rhodium ions but not other divalent cations. Strains containing null alleles of COT1 are viable yet more sensitive to cobalt than are wild-type strains. Transcription of COT1 responds minimally to the extracellular cobalt concentration. Addition of cobalt ions to growth media results in a twofold increase in COT1 mRNA abundance. The gene encodes a 48-kDa protein which is found in mitochondrial membrane fractions of cells. The protein contains six possible membrane-spanning domains and several potential metal-binding amino acid residues. The COT1 protein shares 60% identity with the ZRC1 gene product, which confers resistance to zinc and cadmium ions. Cobalt transport studies indicate that the COT1 product is involved in the uptake of cobalt ions yet is not solely responsible for it. The increased tolerance of strains containing multiple copies of the COT1 gene is probably due to increased compartmentalization or sequestration of the ion within mitochondria.


[Immunoelectron-microscopic determination of the localization of tryptophanyl-tRNA synthetase in eubacterial cells of Escherichia coli and Methanococcus halophilus archaebacteria].

Localization of tryptophanyl-tRNA-synthetase (TRS) was studied in halophilic archaebacterium Methanococcus halophilus and eubacterium E. coli. Ultrathin sections of the cells, fixed with glutaraldehyde and embedded in "Lowicryl K4M" at -35 degrees C, were treated with colloidal gold complexes containing monoclonal antibodies Aml against TRS. The latter bind specifically to TRS isolated both from eucaryotes, archae- and eubacteria. According to the label distribution three zones in M. halophilus and E. coli can be distinguished: (i) about 75% of the whole amount of gold particles are localized in the cytoplasm, the distribution of label being more or less homogeneous; (ii) cytoplasmic regions, adjacent to nucleoid, are intensively labelled (about 20% of the whole amount of label); (iii) very few gold particles (not more than 10% of the whole amount) are present in the nucleoid. The data obtained show, that the distribution of TRS in the nucleoid and cytoplasm of archaebacterium M. halophilus is close to the distribution of TRS, found in E. coli. It supports our previous conclusion that the structural organization of transcription-translation apparatus in methanogen and halophilic archaebacteria is similar to that in eubacteria.


Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.


["DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes].

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.


Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.


Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine.

In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.


95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.


Isolation of rsp-1, a novel cDNA capable of suppressing v-Ras transformation.

Using an expression cloning assay, we have isolated a novel cDNA, referred to as rsp-1, which suppresses the v-Ras-transformed phenotype. When introduced into NIH 3T3 fibroblasts under the control of a metallothionein promoter, rsp-1 confers resistance to v-Ras, but not to v-Mos or v-Src, and inhibits growth of the cells. The rsp-1 cDNA contains a 831-bp open reading frame encoding a 277-amino-acid leucine-rich protein. The rsp-1 cDNA exhibits no significant homology to sequences in the DNA data bases. However, searches of the protein data bases revealed that it contains a series of leucine-based repeats which are homologous to the leucine repeats found in the regulatory region of the yeast adenylyl cyclase. rsp-1 specific RNA is detectable in a wide variety of cell lines and tissues, and the gene is conserved among eukaryotic species. These data suggest that rsp-1 plays a role in Ras signal transduction.


Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.


Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.


Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.


Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.

The src family gene lyn is expressed preferentially in B lymphocytes but very little in normal T lymphocytes. Transcription of the lyn gene in T lymphocytes was shown to be induced by the p40tax protein encoded by human T-cell lymphotropic virus type I. For determination of the mechanism of p40tax-mediated trans activation, the transcriptional promoter region of the lyn gene was characterized. By endonuclease S1 mapping, the transcriptional initiation sites were identified within the 770-bp EcoRI-SacI fragment of the 5'-terminal portion of the human lyn gene. This fragment showed promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into various cell lines. Nucleotide sequence analysis revealed that the lyn promoter region contained four GC box-like sequences but not a TATA or CCAAT box. In addition, it contained sequences characteristic of a cyclic AMP-responsive element, octamer-binding motif, PEA3-like motifs, and NF kappa B-binding motif-like sequence. Mutational analysis suggested that the octamer-binding motif sequence is of primary importance for the lyn promoter activity but that the other elements are not. Cotransfection of various chloramphenicol acetyltransferase constructs containing different length of the lyn promoter together with p40tax expression plasmids into Jurkat T cells showed that the sequence responsible for p40tax-induced transcription is present around the transcription initiation sites.


Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1.

We have previously shown that a trans-acting protein produced in some tissue culture cells positively control the transcriptional activity directed by the mouse p12 promoter. This nuclear protein exerts its positive activity by interacting with a regulatory sequence designated p12.A and located between the TATA and CCAAT box elements on the p12 gene promoter. Using DNase I and dimethyl sulfate methylation interference footprinting techniques coupled with gel retardation assays, we found evidence that the protein which binds to the p12.A element is the well-known transcription factor Sp1. Mutational analysis in transient transfection assays confirmed the positive activity exerted by this protein in every cell line tested. In agreement with this observation, we detected a p12.A-Sp1 binding activity in nuclear extracts prepared from all cell lines used. However, a similar binding activity could not be detected in a number of nuclear extracts prepared from normal mouse tissues. In this report, we provide the evidence that the lack of Sp1-binding activity results from the degradation of Sp1 in the kidney, liver, and pancreas of the mouse.


Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.


Role of GCR2 in transcriptional activation of yeast glycolytic genes.

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.


PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.


NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.


Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences.

The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.


Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.


Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.


Growth factor-induced delayed early response genes.

Growth factors induce the sequential expression of cellular genes whose products are thought to mediate long-term responses to the growth factors. In mouse 3T3 fibroblastic cells, the first genes to be expressed (immediate-early genes) are activated within minutes after the addition of platelet-derived growth factor, fibroblast growth factor, or serum. By cDNA cloning, we have identified genes that are activated after a delay of a few hours and several hours prior to serum-induced DNA replication. Activation of these delayed early response genes requires new protein synthesis, presumably the synthesis of immediate-early transcription factors described previously. Partial or complete sequencing of 13 different delayed early cDNAs, representing about 40% of the 650 primary cDNA isolates, revealed that 8 were related to known gene sequences and 5 were not. Among the former are cDNAs encoding nonhistone chromosomal proteins [HMGI(Y) and HMGI-C], adenine phosphoribosyltransferase (APRT), a protein related to human macrophage migration inhibitory factor (MIF), a protein of the major intrinsic protein (MIP) family homologous to the integral membrane protein of human erythrocytes, and cyclin CYL1. In 3T3 cells, the delayed early gene response to growth factors appears to be at least as complex as the immediate-early gene response previously described.


A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.


PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.


Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.


Sequence analysis reveals homology between two proteins of the flagellar radial spoke.

Flagellar radial spokes contribute to the regulation of dynein arm activity and thus the pattern of flagellar bending. We have sequenced the genes for radial spoke protein 4 (RSP4) and RSP6, two of the five proteins that make up the radial spoke head in Chlamydomonas reinhardtii. The two genes, which are tightly linked genetically (B. Huang, G. Piperno, Z. Ramanis, and D.J.L. Luck, J. Cell Biol. 88:80-88, 1981), are separated by only 435 bp. They encode proline-rich polypeptides of 49.8 kDa (RSP4) and 48.8 kDa (RSP6), which are 48% identical to each other but do not resemble any previously sequenced proteins. The transcription start sites of these genes and an additional radial spoke protein gene, that for RSP3, were determined, and patterns of mRNA accumulation during flagellar regeneration were examined for the three radial spoke protein genes. These studies provide the molecular tools for a detailed analysis of radial spoke head function and assembly and for a determination of the mechanism by which the genes required to build a complex organelle are regulated.


Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.


A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.


DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.


Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.


Replication forks pause at yeast centromeres.

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.


Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel.

The cis-acting control elements of the interleukin-2 receptor alpha-chain (IL-2R alpha) gene contain a potent kappa B-like enhancer whose activity can be induced by various mitogenic stimuli. Recent cloning of the p50 and p65 subunits of the kappa B-binding protein NF-kappa B complex revealed a striking sequence homology of these proteins with the c-rel proto-oncogene product (c-Rel). On the basis of this homology, we examined the potential role of c-Rel in controlling IL-2R alpha transcription. We now demonstrate that the recombinant human c-Rel protein binds to the kappa B element in the IL-2R alpha promoter and results in alteration of the DNA structure in the adjacent downstream regulatory elements containing the CArG box and the GC box. We found that human c-Rel can activate transcription from the IL-2R alpha promoter, but not the kappa B-containing human immunodeficiency virus type 1 promoter, upon cotransfection into Jurkat T cells. Furthermore, truncation of the carboxyl terminus of c-Rel results in a c-Rel mutant (RelNA) that (i) localizes exclusively in the nucleus and (ii) acts in synergy with wild-type c-Rel in activating transcription from the kappa B site of the IL-2R alpha promoter. Finally, induction of surface IL-2R alpha expression coincides with the induced levels of endogenous c-Rel and induced c-Rel binding to the IL-2R alpha kappa B site. Our study identified c-Rel as one component of the Rel/NF-kappa B-family proteins involved in the kappa B-dependent activation of IL-2R alpha gene expression. Furthermore, our results suggest that a Re1NA-like cellular factor (e.g., NF-kappa B p50 or p49 subunit) acts in synergy with c-Re1 during T-cell activation.


The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters.

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.


Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.


Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.


Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.


A cyclic AMP-responsive element-binding transcriptional activator in Drosophila melanogaster, dCREB-A, is a member of the leucine zipper family.

In this report, we describe the isolation and initial characterization of a Drosophila protein, dCREB-A, that can bind the somatostatin cyclic AMP (cAMP)-responsive element and is capable of activating transcription in cell culture. Sequence analysis demonstrates that this protein is a member of the leucine zipper family of transcription factors. dCREB-A is unusual in that it contains six hydrophobic residue iterations in the zipper domain rather than the four or five commonly found in this group of proteins. The DNA-binding domain is more closely related to mammalian CREB than to the AP-1 factors in both sequence homology and specificity of cAMP-responsive element binding. In embryos, dCREB-A is expressed in the developing salivary gland. A more complex pattern of expression is detected in the adult; transcripts are found in the brain and optic lobe cell bodies, salivary gland, and midgut epithelial cells of the cardia. In females, dCREB-A is expressed in the ovarian columnar follicle cells, and in males, dCREB-A RNA is seen in the seminal vesicle, ejaculatory duct, and ejaculatory bulb. These results suggest that the dCREB-A transcription factor may be involved in fertility and neurological functions.


Formation of the 3' end of sea urchin U1 small nuclear RNA occurs independently of the conserved 3' box and on transcripts initiated from a histone promoter.

The formation of the 3' end of vertebrate small nuclear RNAs (snRNAs) requires that transcription initiate from an snRNA promoter. There is a loosely conserved required box 5 to 20 nucleotides (nt) 3' of the gene. The sea urchin snRNA genes contain promoter elements different from those of the vertebrate snRNAs. They also contain a characteristic 3' 15-nt sequence which is conserved among different sea urchin snRNA genes. We used microinjection of sea urchin U1 snRNA genes into sea urchin zygotes to define the sequence requirements for U1 snRNA 3'-end formation. Surprisingly, the conserved 3' box is not required for efficient 3'-end formation in vivo. Deletion analysis reveals that the 6 nt immediately 3' of the U1 snRNA are involved in 3'-end formation. Substitution analysis revealed that either these 6 nt 3' of the U1 RNA or the conserved 3' box could direct 3'-end formation. Transcripts initiated from a histone H4 promoter formed U1 3' ends about 50% as efficiently as transcripts initiated from the U1 promoter, even when the U1 end was placed in tandem with a histone 3'-processing signal, suggesting that transcription from an snRNA promoter is not necessary for formation of the 3' end of sea urchin U1 snRNA.


Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.


Identification of an early-growth-response gene encoding a novel putative protein kinase.

Early-growth-response genes, also known as immediate-early genes, play important roles in regulating cell proliferation. We have identified a new type of early-growth-response gene product, a 77,811-Da putative serine/threonine kinase, which is highly inducible by serum and phorbol ester. mRNA encoding this putative kinase is markedly elevated within 1 h after treatment with mitogen, and this induction is synergistically increased by cycloheximide. Dexamethasone blocks serum induction of the kinase mRNA, as does transformation by v-Ki-ras. The kinase mRNA was detected in mouse brain, lung, and heart. This new putative kinase, which we term Snk, for serum-inducible kinase, showed similarity in its proposed catalytic domain to many other protein kinases; however, no other kinase showed enough sequence similarity with Snk to suggest the existence of a common function. Hence, Snk represents a new type of protein kinase involved in the early mitogenic response whose activity is transcriptionally and posttranscriptionally regulated.


Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression.

We previously observed that human ADA gene expression, required for the intrathymic maturation of T cells, is controlled by first-intron sequences. Used as a cis activator, the intron generates copy-dependent reporter expression in transgenic thymocytes, and we here dissect its critical determinants. Of six DNase I-hypersensitive sites (HS sites) in the intron, only HS III was a transfection-active classic enhancer in T cells. The enhancer contains a critical core region, ACATGGCAGTTGGTGGTGGAGGGGAACA, that interacts with at least two factors, ADA-NF1 and ADA-NF2. Activity of the core is strongly augmented by adjacent elements contained within a 200-bp domain corresponding to the limits of HS III hypersensitivity. These core-adjacent sequences include consensus matches for recognition by the AP-1, TCF-1 alpha, mu E, and Ets transcription factor families. In contrast, considerably more extensive sequences flanking the enhancer domain were required for position-independent and copy-proportional expression in transgenic mouse thymocytes. The additionally required upstream segment encompassed the nonenhancer HS II site. The required downstream segment, composed largely of Alu-repetitive DNA, was non-DNase I hypersensitive. Transgenes that lacked either segment were subject to strong positional effects. Among these variably expressing lines, the expression level correlated with the degree of hypersensitivity at HS III. This finding suggests that formation of hypersensitivity is normally facilitated by the flanking segments. These results delineate a complex thymic regulatory region within the intron and indicate that a series of interactions is necessary for the enhancer domain to function consistently within chromatin.


Thymic overexpression of Ttg-1 in transgenic mice results in T-cell acute lymphoblastic leukemia/lymphoma.

T-cell translocation gene 1 (Ttg-1), also called rhombotin, is deregulated upon translocation into the alpha/delta T-cell receptor loci in acute lymphoblastic leukemias bearing the t(11;14)(p15;q11). Ttg-1 encodes a nuclear protein, expressed predominantly in neuronal cells, which belongs to a novel family of transcription factors possessing LIM domains. We utilized the lck proximal promoter to overexpress this candidate oncogene in immature thymocytes of transgenic mice. lckPr Ttg-1 mice develop immature, aggressive T-cell leukemia/lymphomas. Tumor incidence is proportional to the level of Ttg-1 expression. Most tumors contain CD4+8+ cells as well as CD4-8+ cells, which have an immature rather than a mature peripheral phenotype. Ttg-1-induced tumorigenesis preferentially affects a minority population of thymocytes representing an immature CD4-8+ intermediate stage between double-negative CD4-8- cells and double-positive CD4+8+ cells. This model indicates that the aberrant expression of putative transcription factors plays a primary role in the genesis of T-cell acute lymphoblastic leukemias.


Functional antagonism between YY1 and the serum response factor.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.


Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae.

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.


Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Pax genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.


Endothelin induces transcription of fos/jun family genes: a prominent role for calcium ion.

The 21-amino acid mammalian peptide endothelin (ET) is a powerful vasoconstrictor, a mitogen for fibroblasts and vascular smooth muscle cells, and a potent effector for numerous tissues. Through extracellular interaction with G protein-coupled transmembrane receptors, ET stimulates intracellular second messenger events that in turn activate immediate early gene transcription. Using Northern blot hybridization and nuclear run-on analyses, we examined the modulation of c-fos, fos-B, fra-1, c-jun, and jun-B gene transcripts in Rat-1 fibroblasts after ET treatment. Furthermore, we investigated the role that intracellular Ca2+ transients played in effecting this gene regulation, using the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to block Ca(2+)-dependent transcription. Our results demonstrate that ET rapidly effects increased RNA levels for all five fos/jun family genes investigated, at least two of them by increasing gene transcription. Furthermore, our results argue that increased intracellular free Ca2+ is directly involved in the induction of these fos/jun family genes by ET. While mobilization of intracellular Ca2+ is not the only pathway to fos/jun gene induction used by ET, it is clearly a major component of the signaling apparatus that is set in motion by this potent effector.


Expression of prolactin and its receptor in human lymphoid cells.

We have investigated whether human lymphoid cells are able to synthesize and secrete human PRL (hPRL) and to express PRL receptors. Metabolic labeling with [35S]methionine and immunoprecipitation of cell extracts from human mononuclear cells (MNC) and a human T lymphocyte cell line with an antiserum against hPRL revealed protein of M(r) 23,000, identical in size to pituitary hPRL. Dilution curves of lymphocyte immunoreactive hPRL were parallel to those obtained with pituitary hPRL in an immunoradiometric assay using two monoclonal antibodies against hPRL. Polymerase chain reaction experiments with primers located in the coding sequence of hPRL showed that the hPRL gene was expressed in MNC. Furthermore, cDNA cloning and sequence analysis indicated the presence of an extra 5' noncoding exon previously described for decidual hPRL. When MNCs were further separated into B cells, T cells, and monocytes, the expression of hPRL appeared to be mainly associated with the T lymphocyte fraction. The hPRL transcript was also detected in thymocytes and in a set of human lymphoid cell lines. Finally, polymerase chain reaction experiments revealed a ubiquitous distribution of PRL receptor gene expression in B cells, T cells, and monocytes. The presence of the receptor for PRL and production of PRL by T lymphocytes suggest a possible autocrine or paracrine effect of PRL in immune cell function.


Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.


Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast.

The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.


Differential promoter usage by the rat 11 beta-hydroxysteroid dehydrogenase gene.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) catalyzes the conversion of physiological glucocorticoids to inactive products, thus protecting nonselective renal mineralocorticoid receptors from circulating glucocorticoids (ensuring aldosterone selectivity in vivo) and modulating glucocorticoid access to mineralocorticoid receptors and glucocorticoid receptors in other tissues. Detection of multiple mRNA and immunoreactive 11 beta-OHSD species in kidney, but not liver, extracts suggests the presence of tissue-specific isoforms. To determine whether differential promoter usage might explain the mRNA heterogeneity we cloned and sequenced rat 11 beta-OHSD genomic DNA. Total identity was found between the nucleotide sequence of exons 1 and 2 and the previously published rat liver cDNA. Using both primer extension and RNase protection analyses we found the predominant transcription start site in liver (+1) is 105 base pairs (bp)5' of the start of translation. In kidney two additional Cap sites were detected: 1) 264 bp 5' of exon 1; there is no in-phase open reading frame, suggesting the additional 5' sequence is not translated; and 2) 65 bp upstream of exon 2, within intron A; the predicted truncated protein lacks the first 26 hydrophobic residues. Oligonucleotide probes specific to transcripts arising from each promoter confirmed that all three are employed in kidney, whereas a single predominant species was found in liver, thus demonstrating tissue-specific differential promoter usage of the 11 beta-OHSD gene.


Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)