Cloning and sequencing of the aculeacin A acylase-encoding gene from Actinoplanes utahensis and expression in Streptomyces lividans.

Aculeacin A acylase (AAC), produced by Actinoplanes utahensis, catalyzes the hydrolysis of the palmitoyl moiety of the antifungal antibiotic, aculeacin A. Using mixed oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequences of the two subunits of AAC, overlapping clones were identified in a cosmid library of A. utahensis DNA. After the sub-cloning of a 3.0-kb fragment into Streptomyces lividans, the recombinant produced AAC extracellularly. The nucleotide sequence of this fragment predicted an open reading frame of 2358 bp with GTG start and TGA stop codons. The deduced 786-aa sequence should correspond to a single polypeptide chain, indicating that this polypeptide is processed to the active form which is composed of the two subunits. Threefold more AAC was obtained from the S. lividans recombinant carrying the cloned gene than the original A. utahensis strain.


Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.

One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding. The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate. The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx. 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer. The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer. We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands. We obtained the following results: in full-site (FRT) vs. full-site recombinations or in full-site vs. half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant. This difference virtually disappeared when reactions were done with half-FRT sites. We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis.


A multisite integrative cassette for the yeast Saccharomyces cerevisiae.

We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Saccharomyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences. This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sigma elements by gene replacement. Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes. Consequently, these studies led to the genetical tagging of individual members of the sigma family.


Characterization of the tRNA(Trp) genes of Saccharomyces cerevisiae.

The purpose of this work was to examine the tRNA(Trp)-encoding genes (tRNA(Trp)) of Saccharomyces cerevisiae to gain insight as to why tRNA(Trp) amber suppressors, isolated by conventional genetic techniques, have not been reported. The results herein indicate that the haploid yeast genome contains six tRNA(Trp) genes which map to five or six chromosomes. Not only do the six genes have identical coding sequences but their introns are also identical. Gene replacement experiments indicate that five copies of tRNA(Trp) are sufficient for cell viability. Thus, mutation of one tRNA(Trp) gene to a suppressor in vivo, lowering the functional number of tRNA(Trp) genes, would not be expected to be lethal.


Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.


Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints.

We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant. The rad3 gene was mapped to NotI fragment C on chromosome II.


Stable incorporation of genetic material into the chromosome of Rhizobium meliloti 41: construction of an integrative vector system.

An integrative vector system has been developed from the site-specific recombination elements of temperate phage 16-3. The system can be used for highly efficient stable introduction of genetic material into the chromosome of the symbiotic nitrogen-fixing organism, Rhizobium meliloti 41 (Rm41) at the attB site. Vectors carrying the phage-borne attachment site were constructed, and helper phages providing the site-specific recombination functions in trans were isolated. Other possible applications of the system are discussed.


Stabilization of T7-promoter-based pARHS expression vectors using the parB locus.

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct the pARHS vectors. These vectors are stable for at least 60 cell generations, even in the absence of selection by an antibiotic present in the culture media, both with or without IPTG induction.


Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii.

Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guanosine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).


Cloning, sequence and expression of a beta-tubulin-encoding gene in the homobasidiomycete Schizophyllum commune.

The beta-tubulin (beta Tub)-encoding gene (tub-2) of Schizophyllum commune is the first tubulin gene isolated, cloned and sequenced from higher filamentous fungi (homobasidiomycetes). The S. commune tub-2 gene is organized into nine exons and eight introns. The introns vary from 48 to 107 nt in length, and are distributed throughout the gene. The tub-2 exons code for a protein of 445 amino acids (aa), which shows great homology with beta Tubs of filamentous ascomycetes, plants, and animals, but less homology with yeasts. The codon usage of tub-2 from S. commune is biased, as it is in most beta Tub-encoding genes of filamentous fungi. The S. commune beta Tub shows a conserved aa sequence in the C-terminal domain, which is suggested to interact with microtubule-associated proteins in animals. In contrast, the S. commune beta Tub deviates from most known beta Tubs by having a Cys165 residue, which might be significant for the insensitivity of S. commune haploid strains to the antimicrotubule drug, benomyl. In tub-2 of different haploid strains, sequence polymorphisms occur in the 5' and 3' flanking regions. The expression of tub-2 is high in young mycelium, which has a high number of extending apical cells, but decreases with the aging of the mycelium. No significant difference in the hybridization signal intensity for the tub-2 transcripts was recorded either during intercellular nuclear migration at early mating, or in mycelia with a mutation in the B mating-type gene.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation and characterization of a cellulose-growth-specific gene from Agaricus bisporus.

The edible basidiomycete, Agaricus bisporus, produces extracellular endoglucanase. Endoglucanase production is induced by cellulose and repressed by fructose in A. bisporus grown on minimal medium, and is regulated in activity during fruiting body development. An anti-endoglucanase antibody was used to isolate cellulase-related genes. Three main polypeptides of 38, 58, and 60 kDa were immunoprecipitated by the antibody from products of in vitro cell-free translation of mRNAs isolated from cellulose-grown mycelium. No cross-reaction was detected with the translated products from fructose-grown mycelium. This antibody was used to immunoscreen a lambda ZAPII-cDNA expression library made from mRNA isolated from cellulose-grown mycelium. Two cDNA cross-reacting clones, pSRc110 and pSRc200, were isolated. Clones pSRc110 and pSRc200 cross-hybridized and had the same restriction map. Clone pSRc200 hybrid selected an mRNA that on cell-free translation produced a 38-kDa polypeptide. The cDNA fragment from pSRc200 hybridized to a 1.3-kb mRNA from cellulose-grown mycelium. No hybridization was observed when using fructose-grown mycelium mRNA. Thus, the gene (cel1) expressing the 1.3-kb mRNA, is differentially regulated by the carbon source of the culture medium. The cell gene was isolated in a 8.9-kb EcoRI genomic fragment after hybridization to pSRc200. Sequences similar to those in the egl1 and cbh2 genes from Trichoderma reesi were found upstream from the ATG start codon in cel1. Nine short intervening sequences disrupt the cel1 coding sequence, and a strong bias against codons ending with G and A was observed.(ABSTRACT TRUNCATED AT 250 WORDS)


Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae.

We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychia lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M(r) of 16,819. Both ends of the cloned gene have the hypotrichous telomeric C4A4 repeat. The coding region is flanked by a 158-bp 5'-leader sequence and a 3'-trailer sequence of 213 bp. S1 analysis was used to locate the transcription start point (tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca(2+)-binding protein with only one Ca(2+)-binding site. It's function and biological significance is yet unknown.


Expression of a downstream gene from a bicistronic transcription unit in transgenic tobacco plants.

We have constructed a set of plasmids carrying an artificial compact stop-start codon sequence, TGATGTAACATGA, between an upstream open reading frame, terminating at one of the stop codons, and a downstream kanamycin-resistance (KmR)-encoding gene (nptII) initiating at the second ATG. These plasmids were introduced into tobacco protoplasts by direct gene transfer. The efficiency of expression of the downstream nptII gene was measured by scoring the number of KmR transformants. With a closer distance between the functional stop and start codons, a tendency to less efficient expression of nptII was found. The integration and expression of both genes as a bicistronic transcription unit were verified by Southern- and Northern-blot analyses. A possible application of the compact stop-start codon sequence for insertional mutagenesis is discussed.


Compositional bimodality and evolution of retroviral genomes.

The compositional distributions of genomes, genes (and their third codon positions) and long terminal repeats from retroviruses of warm-blooded vertebrates are characterized by a striking bimodality which is accompanied by a remarkable compositional homogeneity within each retroviral genome. A first, major class of retroviral genomes is GC-rich, whereas a second, minor class is GC-poor. Representative expressed viral genomes from the two classes integrate in GC-rich and GC-poor isochores, respectively, of host genomes. The first class comprises all oncoviruses (except B-types and some D-types), the second, lentiviruses, spumaviruses, as well as B-type and some D-type oncoviruses (e.g., mouse mammary tumor virus and simian retroviruses type D, respectively). The compositional bimodal distribution of retroviral genomes and the accompanying compositional homogeneity within each retroviral genome appear to be the result of the compositional evolution of retroviral genomes in their integrated form.


The structure and complete sequence of the gene encoding chicken ribosomal protein L5.

The nucleotide (nt) sequence of the gene encoding chicken ribosomal protein L5, which associates with 5S rRNA, was determined. The gene contains eight exons and seven introns which spread over 7252 bp. The transcription start point (tsp) is a C residue in a tract of 13 pyrimidines, and its 5'-flanking region lacks canonical TATA and CAAT boxes. The G+C content of the region around the tsp is calculated as high as 78%, and nine GC boxes exist within the 5'-flanking region and the first intron. The nt sequence at positions -36 to -22 is similar to an internal sequence at positions 56-70 of the gene encoding chicken 5S rRNA, which corresponds to the transcriptional internal control region of the 5S rRNA-encoding gene of Xenopus laevis. This region might contribute to the coordinate expression of the genes encoding protein L5 and 5S rRNA.


Cloning and expression analysis of two ZFY-related zinc finger genes from Alligator mississippiensis, a species with temperature-dependent sex determination.

In order to investigate the molecular mechanism of temperature-dependent sex determination, a human zinc finger gene (ZFY), known to be highly conserved amongst other species, was used to isolate homologues from the genome of the American alligator, Alligator mississippiensis. ZFY was originally a candidate for the primary testis-determining gene in man, but is now thought to function further down the sex-determining cascade. Two alligator genes are described, Zfc and Znc6. Both code for zinc finger proteins and exhibit amino acid (aa) homologies to ZFY of 91% and 73%, respectively. Znc6 shows aa homology of 88% to the protein encoded by the zinc finger exon of the human ZFY-related gene, ZNF6, recently found on the X chromosome. Analysis of Zfc and Znc6 expression during embryonic development identified two major transcripts of 5.9 kb and 2.7 kb coding for Zfc, whilst only one transcript of 4.8 kb was detected for Znc6. Both genes are transcribed at all stages tested, from day 3 (post egg laying) throughout gestation. The expression level of all transcripts appears to decline towards the time of hatching (65-72 days). No sex-specific differences in the expression were observed. The extensive sequence conservation of the genes between reptiles and humans suggests major functional constraints. The expression patterns indicate that these genes do not play a primary role in temperature-dependent sex determination.


A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor.

A CArG box motif [CC(A+T-rich)6GG] is one of the DNA elements required for muscle-specific gene transcription. Nuclear factors in mouse C2 myogenic cells strongly bind to the CArG box in the first intron of the gene (Sm alpha-A) encoding human smooth muscle alpha-actin. To clone cDNAs of the CArG box-binding factor (CBF), lambda gt11 cDNA expression libraries from C2 cells were screened for in situ DNA binding specific for this CArG box sequence. The 1.6-kb cDNA (CBF-A) encoding 285 amino acids (aa) was obtained, and a beta-galactosidase fusion protein, bacterially produced from the cDNA, bound to DNA fragments containing several CArG boxes. When the expression level of CBF-A in C2 cells increased by transfection of CBF-A expression plasmids, Sm alpha-A transcription was repressed. The deduced aa sequence of CBF-A is similar to some single-stranded (ss) nucleic acid-binding proteins. The fusion protein could bind to ssDNA, whereas CBF in C2 cell nuclear extracts could not. From these results, CBF-A is a novel CArG box-, ssDNA- and RNA-binding protein, as well as a repressive transcriptional factor.


Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast.

We have constructed yeast strains in which the expression of the Saccharomyces cerevisiae URA3 gene is induced by the human estrogen receptor (hER). Promoter sequences required for both basal and activated transcription of URA3 were replaced with one or three estrogen-response elements (EREs) positioned upstream of the native TATA box. These constructs were each integrated at the TRP1 locus of a yeast strain in which the natural URA3 gene had been deleted, and the integrants were transformed with low- or high-copy-number shuttle plasmids expressing wild-type or truncated derivatives of hER. Transformants were assayed for growth on uracil-deficient medium plus or minus estradiol (E2), for resistance to 5-fluoroorotic acid (5-FOA) and for activity of OMPdecase (orotidine-5'-monophosphate decarboxylase), the product of the URA3 gene. We show that the growth and 5-FOA-resistance (5-FOAR) phenotypes of these strains are strictly dependent upon the function of the receptor derivatives. Induction of URA3, measured by OMPdecase activity, was observed over a 20- to 2500-fold range depending on the receptor derivative, its expression level and the number of EREs in the responsive promoter. Both one- and three-ERE reporter strains expressing the full-length receptor are completely E2-dependent for growth, and display a 5-FOAR phenotype in the absence of the hormone. We demonstrate that the individual hER transactivation functions, TAF1 and TAF2, are both functional in yeast, and that the hormone-dependent TAF2 is the more potent activator on our reporters. We show that hER displays strong homosynergism in yeast, and discuss the contributions of the two TAFs in hER synergism.(ABSTRACT TRUNCATED AT 250 WORDS)


Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii.

Several group-I introns have been shown to specifically invade intron-minus alleles of the genes that contain them. This type of intron mobility is referred to as 'intron homing', and depends on restriction endonucleases (ENases) encoded by the mobile introns. The ENase cleaves the intron-minus allele near the site of intron insertion, thereby initiating gene conversion. The 23S (LSU) rRNA-encoding gene (LSU) of the chloroplast genome of Chlamydomonas reinhardtii contains a self-splicing group-I intron (CrLSU) that has a free-standing open reading frame (ORF) of 163 codons. Translation of CrLSU intron RNA in cell-free systems produces a polypeptide of approx. 18 kDa, the size expected for correct translation of the ORF. The in vitro-synthesized 18-kDa protein cleaves plasmid DNA that contains a portion of LSU where the intron normally resides, but lacking the intron itself. Cleavage by the intron-encoded enzyme (I-CreI) occurs 5 bp and 1 bp 3' to the intron insertion site (in the 3'-exon) in the top (/) and bottom (,) strands, respectively, resulting in 4-nt single-stranded overhangs with 3'-OH termini. We also show that the recognition sequence of I-CreI spans the cleavage site and is 24 bp in length (5'-CAAAACGTC,GTGA/GACAGTTTGGT).


Amplification and characterization of an inverted repeat from the Chlamydomonas reinhardtii mitochondrial genome.

Based on the nucleotide (nt) sequences of cob and L2a, two oligodeoxyribonucleotides (oligos) were synthesized and used in the polymerase chain reaction (PCR) to amplify the termini of the Chlamydomonas reinhardtii mitochondrial (mt) genome. A 0.8-kb PCR product was detected by agarose-gel electrophoresis when using unligated mt DNA as the template for PCR. This may have indicated the presence of a naturally occurring circular mt DNA molecule that acted as the PCR template. The 0.8-kb DNA could also be amplified from the linear mt DNA via an intramolecular jump during PCR. The sequence data from the 0.8-kb PCR product, and the right 0.6-kb and left 1-kb terminal fragments of the linear mt DNA, along with Southern hybridization analysis, indicated that a 0.49-kb inverted repeat (IR) sequence is present at the right and left termini of the linear mt DNA. The IR contains A+T-rich clusters, as well as numerous short direct repeats (DR) and IR, and might be involved in the recombination, replication and expression of the C. reinhardtii mt genome.


Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast.

The unprocessed HTLV-I Gag precursor, Pr53gag, was synthesized in yeast, Saccharomyces cerevisiae. The synthesized Pr53gag was myristoylated, associated with the cellular membrane, and released into the culture medium. The released Pr53gag was pelleted by centrifugation at 100,000 x g for 2 h. Conversion of Gly2 to Ala allowed synthesis of a non-myristoylated soluble Pr53gag which was not released into the culture medium. These results suggest that the release of the HTLV-I Gag precursors Pr53gag, occurs in yeast in a myristoylation-dependent manner.


Four synonymous genes encode calmodulin in the teleost fish, medaka (Oryzias latipes): conservation of the multigene one-protein principle.

We cloned four distinct calmodulin (CaM)-encoding cDNAs from a small teleost fish, medaka (Oryzias latipes). The deduced amino acid (aa) sequences were exactly the same in these four genes and identical to the aa sequence of mammalian CaM, because of synonymous codon usages. The four cDNAs from medaka, termed CaM-A, -B, -C and -D, corresponded to mRNAs of 1.8, 1.4, 2.5 and 1.8 kb, respectively, in Northern blot analysis. Our results demonstrated that the 'multigene one-protein' principle of CaM synthesis is applicable to medaka, as well as to mammals whose CaM is encoded by at least three different genes.


The pyrimidine sequence encompassing the transcription start point of Xenopus laevis ribosomal-protein-encoding genes is not obligatory for activity in oocytes.

The genes coding for ribosomal proteins (r-proteins) in Xenopus laevis have a stretch of about 20 pyrimidines (Y) at their 5' end, in the middle of which is localized the main transcription start point (tsp). To obtain information about its possible functional significance, we have introduced by site-directed mutagenesis one or more purines at various positions within the oligo(Y) tract present at the 5' end of the gene coding for r-protein S19 of X. laevis. The effect of these mutations on transcription and translation of a reporter-coding sequence has been evaluated by DNA microinjection in X. laevis oocytes. We show that an uninterrupted stretch of pyrimidines is not necessary for efficient transcription and translation of the reporter gene in the X. laevis oocyte. This finding does not exclude the possibility that this sequence is involved in transcription and/or translation regulation in some developmental situations different from oogenesis.


Isolation and characterization of the mouse alpha-lactalbumin-encoding gene: interspecies comparison, tissue- and stage-specific expression.

The murine alpha-lactalbumin-encoding gene (m alpha La) was isolated and completely sequenced. The 2.3-kb transcription unit shared a similar organization with that of its counterparts from other species. Sequence comparison for the proximal 5'-flanking region indicated the presence of a consensus motif that occurs in all milk-protein-encoding genes, except the kappa-casein-encoding gene. This may correspond to the binding site for the recently identified mammary-gland-specific factor. The m alpha La gene occurs in a single copy per haploid genome and is specifically expressed in the mammary gland where it is induced during late pregnancy.


A silencer-like cis element for the testis-specific phosphoglycerate-kinase-2-encoding gene.

Phosphoglycerate kinase (PGK), a glycolytic enzyme, possesses two isozymes: somatic-type PGK-1 and testis-specific PGK-2, encoded by distinct genes. Tissue-specific expression of the two PGK-encoding genes (Pgk) seems to be transcriptionally controlled, since tissue distribution of the mRNAs coincides well with that of the proteins. In the present study, we determined the cis-acting DNA elements that regulate the transcription of mouse Pgk-2. A transient expression assay of DNAs having various portions of the Pgk-2 upstream region linked to the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) was performed using mouse cell lines that exclusively express Pgk-1. A substantial increase in cat expression was observed when the region between nucleotides (nt) -1404 and -685, relative to the most distal transcription start point at nt +1, was lost. This cis-acting region appeared to function as a silencer, since it repressed cat expression independently of either orientation to or distance from the Pgk-2 promoter. Moreover, the cis element inhibited Pgk-2 transcription with no effect on Pgk-1 transcription in a cell-free system using nuclear extracts of rat liver. These results suggest that a silencer-like negative cis element is responsible, at least partly, for tissue-specific transcription of Pgk-2.


Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.


Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147.

Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible D,D-carboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.


Characterization of the ilv-2 gene from Neurospora crassa encoding alpha-keto-beta-hydroxylacyl reductoisomerase.

We have isolated the cDNA and corresponding genomic DNA for the ilv-2 locus of Neurospora crassa. This gene encodes alpha-keto-beta-hydroxylacyl reductoisomerase (Ilv-2), required for the synthesis of isoleucine and valine. The gene contains four introns, maps to the right arm of chromosome V, and encodes a protein of 400 amino acids (aa). Alignment of the aa sequence of Ilv-2 with the two other known eukaryotic sequences encoding this enzyme reveals two conserved regions.


Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae.

The nucleotide (nt) sequence of a starvation-stress gene (ssgA) of the entomopathogenic fungus, Metarhizium anisopliae, and its deduced amino acid (aa) sequence were determined. The primary structure of the SSGA (96 aa; deduced M(r) = 9925; pI = 4.1) protein shares extensive similarities with fungal wall proteins of the 'hydrophobin' class, and the eight Cys residues and putative signal sequences are conserved. Secondary structure predictions suggest an additional resemblance to low-M(r) toxins and agglutinins. Northern (RNA) blot analysis and nuclear run-on assays demonstrated transcriptional control of expression of ssgA during nutrient deprivation and during formation of infection structures. Hybridizations of M. anisopliae genomic DNA indicate that there is only one form of ssgA in the genome.


Sequence of the complete osp operon encoding two major outer membrane proteins of a European Borrelia burgdorferi isolate (B29)

The nucleotide sequence of the operon encoding the major outer surface proteins, OspA and OspB, of a European isolate of Borrelia burgdorferi (strain B29) was determined and compared to the osp operon of the American strain, B31. An amino acid (aa) identity of 80.7% was found when comparing the OspA of B29 with that of B31, whereas the aa sequence of the OspB of B29 reveals only 61.3% identity with the OspB of B31. Thus, strains B31 and B29 can be regarded as representatives of different B. burgdorferi groups.


Complete sequence of the major outer membrane protein-encoding gene of Chlamydia trachomatis serovar Da.

The complete nucleotide sequence of the gene omp1 encoding the major outer membrane protein (MOMP) of Chlamydia trachomatis serovar Da was determined following amplification by the polymerase chain reaction. The most closely related complete sequence published to date is that encoding the C. trachomatis L1 MOMP. When the L1 and Da MOMP deduced amino acid (aa) sequences were compared, 16 single-aa differences located mostly in the variable domains I, II, and IV were detected. These regions contain the B-cell epitopes. Additional differences were found in the constant domains I and II, thought to participate in the T-cell response.


Protein V, a novel type-II IgG receptor from Streptococcus sp.: sequence, homologies and putative Fc-binding site.

We have cloned and sequenced the Fc-receptor-encoding gene, fcrV, from a group G streptococcus. Considerable similarity was revealed between the FcRV, FcRA76 and M proteins of group A streptococci in their signal sequences and 3' termini, and between the Fc-binding regions of FcRV and FcRA76. The promoter and terminator regions showed no homology with those of the fcrA76 and M protein-encoding genes. The A1-A4 domains of FcrV (protein V) exhibit a heptapeptide repeat motif which is characteristic of alpha-helical coiled-coil proteins. The sequence, Ser-Asn-Arg-Ala-Ala, in the outer position, 'f' of each domain is highly conserved and may be involved in FcR-IgG interactions.


The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms.

Nucleotide (nt) sequences encoding the elongation factor Tu (EF-Tu), tRNA(Thr) and tRNA(Trp) from Chlamydia trachomatis have been determined. The environment of the EF-Tu-encoding gene (tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus. The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies. Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies. Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root. The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences. Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein.


The cell division cycle gene CDC60 encodes cytosolic leucyl-tRNA synthetase in Saccharomyces cerevisiae.

The cdc60 mutation (for cell division cycle) of the yeast, Saccharomyces cerevisiae, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature [Bedard et al., Curr. Genet. 4 (1981) 205-214]. We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype. Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurospora crassa, implying that CDC60 encodes the corresponding yeast protein. Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle. Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA. The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.


Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.


Differential expression of the invertase-encoding SUC genes in Saccharomyces cerevisiae.

Invertase (INV) is encoded in Saccharomyces cerevisiae by a family of genes, comprising SUC1-SUC5 and SUC7. Production of INV is highly variable, dependent on the strain and SUC gene present in the cell. The differences in INV production derive from the structure of the genes or are dependent on the genetic background of the strain. Centromeric plasmids (based on YCp50) carrying one of the SUC genes (except SUC7) were introduced into a strain (SEY2101) lacking SUC genes. The INV produced by the transformants was dependent on the individual SUC genes, and correlated with INV mRNA levels. Plasmids in which SUC2 had been placed under control of promoters from the other SUC genes, were used to transform SEY2101 cells. The amounts of INV produced by cells carrying hybrid SUC genes were in agreement with the levels expected if the promoter controlled the expression of the SUC2 structural region. It is suggested that the differences in expression are a function of the transcription efficiency of the different SUC gene promoters, based on the divergence of 5' sequences.


An upstream activating sequence from the Aspergillus nidulans gpdA gene.

Introduction of a previously identified promoter element of the Aspergillus nidulans gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase), the so-called gpd box, into the upstream region of the highly regulated A. nidulans amdS gene (encoding acetamidase), significantly increased (up to 30-fold) the expression of the lacZ reporter gene fused to these expression signals. This increase was dependent on the orientation of the gpd box and on the site of introduction into the amdS upstream region. The presence of additional gpdA sequences which flank the gpd box reduced or even extinguished positive effects of the gpd box. omega-Amino acid and carbon catabolite regulation of the amdS promoter were retained after introduction of the gpd box, indicating that the gpd box does not abolish interactions of the regulatory proteins, AmdR and CreA, with the amdS transcription control sequences. Based on the results, it is suggested that the gpd box comprises at least two separate activities: one being orientation dependent, but relatively independent of position of the gpd box in the upstream region, and the other is only functional near other sites of transcriptional control. Most likely, both activities are not involved in regulation of the amdS promoter.


The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)


Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants.

A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 [Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp. 49-78]. This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host). Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.


Derivation of a mathematical expression useful for the construction of complete genomic libraries.

We present and derive a formula that is useful for the design and evaluation of gene cloning experiments in which a complete gene library of the entire genome of an organism is desired. The formula n = ln(1-phi f)/ln(1-f) (in which n is the number of recombinant clones required to ensure a probability, phi, of obtaining at least one of each of all possible gene sequences, and f is the fraction of the genome contained in an average-sized DNA fragment) applies to construction of libraries, in which at least one copy of all the genetic information of a genome is required. The use of this formula for quantitative evaluation of genomic libraries should give greater assurance that a given library would be complete.


Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli.

The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level. The order of conservation at the nt and aa level is rpsL greater than tufA greater than rpsG greater than f usA. A striking difference is that the rpsG-encoded ribosomal protein, S7, in E. coli K-12 is 23 aa longer than in S. typhimurium. The very low (0.18) codon adaptation index of this part of the E. coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension. A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S. typhimurium is noted. In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events. An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.


Acanthamoeba castellanii RNA polymerase II transcription in vitro: accurate initiation at the adenovirus major late promoter.

We have developed and characterized an efficient in vitro system from the protozoan, Acanthamoeba castellanii, that accurately initiates transcription from the adenovirus-2 major late promoter (AdMLP). Transcription by A. castellanii RNA polymerase II (pol II) is initiated at the same nucleotide (nt) that is used by HeLa extracts and is dependent upon adenovirus sequences located between nt -51 and the region around the transcription start point (tsp). The results suggest that the A. castellanii transcription factors for pol II which determine the tsp and the promoter elements that they recognize have been functionally conserved during evolution.


The flax ribosomal RNA-encoding genes are arranged in tandem at a single locus interspersed by 'non-rDNA' sequences.

The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.


Evolution and differential expression of the (1-->3)-beta-glucan endohydrolase-encoding gene family in barley, Hordeum vulgare.

The (1-->3)-beta-D-glucan glucanohydrolases [(1-->3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1-->3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1-->3,1-->4)-beta-glucan 4-glucanohydrolases [(1-->3,1-->4)-GGH; EC 3.2.1.73] further indicate that the (1-->3)-GGHs and (1-->3,1-->4)-GGHs are derived from a single 'super' gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1-->3,1-->4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1-->3,1-->4)-beta-glucans that are the major constituents in barley walls, while the (1-->3)-GGHs, which are unable to degrade the plant (1-->3,1-->4)-beta-glucans, can hydrolyse the (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls.


Construction of a chimeric viral gene expressing plum pox virus coat protein.

The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA (cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA [synthesized by in vitro transcription of a double-stranded (ds) cDNA clone], using an ad hoc synthetic oligodeoxynucleotide as primer. The resulting cDNA was rendered ds and cloned into the plasmid, pBluescribe M13+. Transcription of this chimeric construction produced RNA molecules of 1250 nucleotides in length, which were used as messengers in the in vitro protein-synthesizing systems. The major product of this transcript consists of a 36-kDa polypeptide and was identified as the PPV coat protein (CP) by molecular weight estimation and by immunoprecipitation with a polyclonal antiserum to PPV. Transfer of this cDNA via Agrobacterium tumefaciens into plants was successfully performed. Transgenic Nicotiana plants producing the PPV CP were subsequently obtained.


Comparison of the sequence and structure of transcription factor IIIA from Bufo americanus and Rana pipiens.

Amino acid (aa) sequences of transcription factor IIIA (TFIIIA) from the toad, Bufo americanus, and the grass frog, Rana pipiens, were determined by cDNA cloning and DNA sequencing. The 3'-untranslated regions of the cDNAs reveal that the TFIIIA gene polyadenylation signal is ATTAAA, rather than the conventional AATAAA. The B. americanus and R. pipiens proteins share about 60% aa sequence homology with each other and with Xenopus laevis TFIIIA. Although these results indicate that TFIIIA has more sequence variation than other DNA-binding proteins, a number of conserved features are evident and of likely functional significance. These include potential guanine nucleotide-binding sites at arginines in zinc fingers (ZnF) II, V, and IX, acidic residues between metal-coordinating cysteines, and a basic region in the C-terminal tail possibly involved in transcription promotion. Sequence similarity also exists in an aa stretch bridging the ninth ZnF and C-terminal tail of both TFIIIA and the 5S RNA-binding protein, p43. DNase I protection analyses demonstrate that B. americanus and R. pipiens TFIIIA interact with the internal control region (ICR) of the Xenopus borealis 5S RNA-encoding gene (5S) in different manners: the B. americanus interaction is similar to X. laevis TFIIIA, protecting the entire 5S gene ICR (nt +96 to +43) from DNase I digestion, whereas the R. pipiens TFIIIA strongly protects the ICR from nt +96 up to +78 and less strongly from +78 to +43. Possibly accounting for the binding differences observed, R. pipiens and R. catesbeiana oocyte 5S RNAs (and by inference 5S genes) were found to contain a G or U at nt position 50 while B. americanus, X. laevis, and other eukaryotic 5S RNAs have an A in the analogous position (nt 53 in generalized eukaryotic structure).


Determination of the sequence of an expressible cDNA clone encoding ERp60/calregulin by the use of a novel nested set method.

An analysis of the N-terminal sequence of the luminal endoplasmic reticulum protein, ERp60, showed that it was identical to the well-characterized Ca2+-binding protein, calregulin. A full-length, expressible cDNA clone encoding this protein was isolated from a mouse fibroblast cDNA library. A novel nested set strategy for the production of overlapping fragments for DNA sequencing was used to determine the complete nucleotide (nt) sequence of both strands of the ERp60 clone. This method utilizes a series of nonspecific deletion primers in conjunction with a specific site primer to generate the nested set fragments. This procedure possesses several advantages over other nested set techniques, since it does not require (i) the re-cloning of the DNA insert into other vectors, (ii) any prior knowledge of the restriction sites of the nt sequence, or (iii) the transformation and analysis of bacterial subclones. ERp60 has a 17-amino acid (aa) signal sequence and the mature protein contains 399 aa with a calculated M(r) of 46,347.


Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a lambda gt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M(r) of 30,227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosome-specific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95-102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29-38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.


Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases.

Chitinase 1 (Chi1) is the major extracellular chitinase from the hyperparasitic fungus, Aphanocladium album. We determined the complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chi1. The coding region is interrupted by three short introns (55, 53 and 49 bp long). Chi1 is 423 aa long and begins with a stretch of 34 aa not found in the mature protein. The Chi1 sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans. Compared with other chitinases, A. album Chi1 has only two short similarity regions (12 and 8 aa long), which are also found in bacterial, yeast and some plant chitinases.


Sequence comparison of the Caenorhabditis elegans dpy-13 and col-34 genes, and their deduced collagen products.

A 2232-nucleotide sequence spanning the col-34 gene from the nematode, Caenorhabditis elegans, is presented. This gene, which encodes a collagen protein (Clg), is transcribed from right to left with respect to the genetic map, and convergently with the nearby dpy-13 gene which also encodes a Clg. Both col-34 and dpy-13 have 5'-flanking elements in common with each other and also with other nematode Clg-encoding genes (clg). One element, variants of which are shared by col-7, col-19 and dpy-13, is predicted to be a target for a number of regulatory molecules, possibly including the ceh-18 product, a nematode POU-domain protein. The deduced amino acid sequence of Col-34 has a high degree of homology with the Dpy-13 collagen, although there are significant differences. In particular, one region of Dpy-13, which is predicted to have secondary structure different from Col-34, is altered by the recessive dpy-13(e225) mutation.


Cloning and characterization of KM190, a specific satellite DNA family of Drosophila kitumensis and D. microlabis.

The nucleotide sequences of nine clones, pKA191/1-4 from Drosophila kitumensis and pMR190/1-5 from D. microlabis, were determined. They represent a tandemly arranged and highly repetitive satellite DNA family, KM190, which is specific for the two species.


The Xenopus U7 snRNA-encoding gene has an unusually compact structure.

A subclone containing a single Xenopus borealis U7 snRNA-encoding gene has been microinjected into X. laevis oocyte nuclei to examine its expression using [32P]GTP as an in vivo label. Only two U7 snRNA bands were detected after incubation, and subsequent fractionation of the oocyte showed that only the larger transcript is present in the nucleus. The sequence of this functional U7 gene shows that, in addition to the coding region, it contains, in the appropriate locations, the 3'-box and proximal sequence element (PSE) which are typical of Pol II-transcribed snRNA genes. Surprisingly, the Xenopus U7 gene contains two adjacent octamer-binding motifs located only 12 and 24 bp upstream from the PSE, instead of the usual location around 150-200 bp upstream. No other cis-acting elements appear to be present. A 5' deletion analysis shows that the transcription level of this U7 gene remains constant if sequences upstream of the two octamer motifs are removed, yet is undetectable when an additional 34 bp containing both octamers and the PSE are removed. This confirms that the Xenopus U7 gene is the most compact snRNA-encoding gene isolated to date. A comparison of U7 sequences shows there is a much greater conservation in the 5' half of the molecule, which contains sequences that base-pair with target pre-mRNA, than in the 3' half which can form a single stem-loop structure that varies in size.


Structure of the rainbow trout metallothionein A gene.

To investigate the regulation of metallothionein-encoding genes in fish, we have isolated and sequenced the rainbow trout metallothionein-A-encoding gene (tMT-A) by polymerase chain reaction. This gene spans about 1.1 kb, consists of three exons and two introns, and has an A+T-rich 5'-region which contains a TATAAA signal, and two metal responsive elements (MREs). The transcription start point is centered around an A residue 81 nt upstream of the ATG codon.


A high-level expression vector for human cells.

We have developed a vector (pSupexp), for high-level expression of genes, that is dependent on transactivation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) by the HIV-1 transactivator protein, Tat. The foreign gene, expressed under transcriptional control of the HIV-1 LTR, and the tat gene, expressed under transcriptional control of SV40 early promoter, are expressed from the same plasmid. The vector also has the neomycin resistance-encoding gene (neo), with G418 being used as a dominant selection marker for stable expression. We have cloned the bacterial cat gene into pSupexp and measured transient CAT production in human HeLa and A549 cells. Our results indicate that pSupexpCAT expresses about 25- to 68-fold higher levels of CAT activity as compared to other standard SV40- and Rous sarcoma virus-based vectors, and three- to fivefold more activity than the cytomegalovirus-based vector. Immunoprecipitation of the CAT protein also revealed a high level of production in human cells.


Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4.

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.


Regulatory elements for the tissue-specific expression of the rat serine dehydratase-encoding gene.

L-Serine dehydratase (SDH; EC 4.2.1.13), the key enzyme for serine utilization in the rat, is synthesized primarily in the liver. Cis-acting DNA elements required for liver-specific expression of the SDH gene were identified by two approaches: (1) transient expression assays in primary cultured rat hepatocytes, and in rat fibrosarcoma and normal rat kidney epithelial (NRK-52E) cell lines; and (2) in vitro transcription assays with nuclear extracts prepared from rat liver and spleen. Deletion analyses of the 5' flanking sequences of the gene have defined two functionally different regions: (a) a cell-type-specific promoter located between positions -62 and +10, which is sufficient for liver-specific expression; and (b) a distal promoter region between bp -133 and -63 containing positive cis-acting elements that regulate the promoter activity in a non-tissue-specific fashion. No other cis-acting elements essential for liver-specific expression were found in the region of -134 to 2.1 kb upstream relative to the cap site of SDH.


Sequences for two cDNAs encoding Arabidopsis thaliana eukaryotic protein synthesis initiation factor 4A.

Two distinct cDNAs encoding protein synthesis initiation factor 4A (eIF-4A) were isolated from an Arabidopsis thaliana cDNA library and sequenced. The deduced amino acid sequences from the two cDNAs were compared to eIF-4A from tobacco, mouse and Saccharomyces cerevisiae. The putative ATP-binding sites and RNA helicase motifs were identified.


Sequence of a cDNA encoding the alpha-subunit of wheat translation elongation factor 1.

A cDNA encoding the alpha-subunit of wheat protein synthesis elongation factor 1 (EF-1 alpha) was isolated from a wheat cDNA expression library and sequenced. The deduced amino acid sequence is compared to EF-1 alpha from other species and to elongation factor Tu (EF-Tu) from Escherichia coli. Putative GTP-binding sites are identified.


Sequence analysis of a second cDNA encoding Atlantic salmon (Salmo salar) serum albumin.

Two similar, but distinct, cDNAs for Atlantic salmon serum albumin have been isolated from the same salmon liver. Comparison between the asSA-1 and asSA-2 sequences reveals 1% overall sequence difference.


Cloning and sequencing of a cDNA encoding mouse stromelysin 1.

A cDNA encoding mouse stromelysin 1 was cloned and the 1740-bp sequence was determined. The deduced amino acid (aa) sequence was compared with stromelysin 1 sequences of other mammals. Comparison with a previously published incomplete aa sequence of mouse stromelysin 1 revealed three single aa differences.


A rabbit AldA pseudogene derived from a partially spliced primary aldolase A transcript.

The entire AldA processed pseudogene of rabbit was isolated and characterized. The pseudogene encodes the C-terminal portion of the protein from amino acids (aa) 126-363. There are deletions, insertions and nucleotide (nt) substitutions distributed throughout the 931 bp of identity shared with the 1.4-kb mRNA. There are 21 replacement codon substitutions, including a clearly deleterious change in the stop codon. This processed pseudogene has several uncommon features: (i) it has a 5'-boundary coincident with an intron/exon junction and does not encode the entire mRNA, (ii) there is a broken direct repeat that overlaps the region of shared identity with the mRNA rather than flanking it, and (iii) there is no poly(A) sequence. This processed pseudogene probably arose by integration of a DNA copy of a partially spliced primary transcript. The structure of this gene has added implications for the timing of posttranscriptional processing events.


Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein.

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793-1798] and Ran [Bischoff and Ponstingl, Proc. Natl. Acad. Sci. USA 88 (1991) 10830-10834; Nature 354 (1991) 80-82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347-360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.


[Theodor Kocher: surgery and ethics].

Theodor Kocher's (1841-1917) creative participation in the rise of modern surgery and his internationally prominent standing therein at the turn of the century are briefly outlined. Kocher experienced, however, the last decade of his career as a period of transition. First new developments within medicine and surgery questioned some of the new "radical" operations by then seen as sole progressive therapeutic possibilities. Second, new emotional and ethical conflicts ensued for the solution of which the traditional ethos (viz. "to help and to do no harm") was felt to be insufficient: The choice between two therapeutic possibilities and the unjustice of socially stratified treatment. Kocher tried to cope with this transition scientifically by promoting basic research, emotionally and ethically by explicitly stressing his long familiar Christian faith.


The emergence of endocrinology.

Endocrinology was recognized as a new branch of biological science mainly as a result of events which took place between about 1890 and 1905, but ideas and discoveries dating from antiquity contributed to it also. Experiments supporting the concept of internal secretions by the testicles were described by Aristotle (4th c. B.C.) and by Hunter (18th c.) and Berthold (19th c.). In 1855 Bernard described glucose as an internal secretion of the liver and Addison reported the effects of adrenal disease in man. Adrenalectomy was fatal in animals. Goitre was known in antiquity and cretinism had been described by Paracelsus. Myxoedema was reported by Gull in 1873, and Kocher described cachexia strumipriva in 1883. In 1888 cretinism, myxoedema and cachexia strumipriva were attributed to thyroid insufficiency. In the 1890s Gley found that tetany after thyroidectomy was due to removal of the parathyroids. In 1884 Rehn proposed that toxic goitre was due to thyroid excess. In 1889 Brown-Séquard claimed that injections of testicular extract rejuvenated the elderly, and in 1893 he introduced organotherapy. In 1891 Murray treated myxoedema successfully with thyroid extract. In 1893 Oliver and Schäfer found that an adrenal extract raised the blood pressure, and soon adrenaline was extracted from the adrenal medulla. Adrenocortical deficiency was proposed as the cause of Addison's disease, and in 1896 Osler prepared an extract which relieved one patient. Diabetes mellitus, described in the first century, was usually fatal. Thirst and polyuria followed experimental pancreatectomy, and pancreatic lesions were found in some human diabetics. In the 19th century workers in France and Germany found that diabetes resulted from absence of an internal secretion by the islets of Langerhans and, in 1893, Laguesse described the function of the islets as "endocrine". In 1895 Beatson treated advanced breast cancer successfully by oöphorectomy. In 1895 Schäfer commended study of the internal secretions to physiologists. In 1902 Bayliss and Starling discovered secretin, a chemical messenger secreted by the intestinal mucosa. In 1905 Starling proposed the name "hormone" for this class of internal secretions. By then endocrinology had been launched as a new branch of science. The crucial events which led to the recognition of endocrinology as a new branch of biological science took place between about 1890 and 1905. Many ideas and discoveries dating from antiquity and apparently unrelated at first had, however, contributed to it. Most of the organs and tissues that form the endocrine system were recognized over 100 years ago.(ABSTRACT TRUNCATED AT 400 WORDS)


[The research of Theodor Kocher on the etiology of osteomyelitis and acute struma].

In 1879, Kocher demonstrated experimentally that osteomyelitis is an infectious disease which is caused by a non-specific microorganism (the "micrococcus"). He showed that the infection of the bone marrow is the consequence of a hematogenous dissemination of a local infection, either cutaneous or mucous. The clinical and pathological form of the disease depends not only on the virulence of the microorganism, but also on the state of the tissue in which it develops. In the same year, Kocher suggested that acute strumitis (inflammation of the goitre) has the same aetiology; he did not, however, produce any experimental evidence for this. Thus, Kocher defined in an original way infection as the result of an interaction between a microorganism and his host.


[Theodor Kocher and the beginnings of radiology in Switzerland].

Already in January 1896, Kocher ordered his first clinical radiography. It was executed by Aimé Forster, professor of physics at the University ob Berne. Together, Forster and Kocher elaborated the plans for the Röntgen Institute of the Inselspital, which opened on January 1st, 1898.


[Theodor Kocher and Zurich].

Theodor Kocher was linked to Zurich by many relationships. It was in this town that he spent his last semester at university, and his contacts with his teachers Friedrich Horner and Theodor Billroth were of some importance with regard to his election as a professor at Berne. Kocher was on friendly terms with his Zurich colleagues Rudolf Ulrich Krönlein and Ferdinand Sauerbruch. In spite of many common views, however, the two Swiss surgeons Kocher and Krönlein were not always of the same opinion.


[Theodor Kocher and César Roux].

96 letters addressed by César Roux (1857-1934), the most prestigious surgeon from the Canton of Vaud, to his fiancée, Anna Begoune, native of Russia, allow us to evoke Roux's training in Kocher's department of surgery. The letters shed light on Roux's relationship with Theodor Kocher, whom he compares successively with Billroth and von Volkmann.


[The inclusion of Theodor Kocher in the work of the Trieste surgeon Gustavo Usiglio on thyroid gland tumors (1894)].

Gustavo Usiglio (1855-1933), a pupil of Billroth, was a surgeon of high qualities. The present author analyzes his main scientific work "On the tumours of the thyroid and their cure"; the book, which deals essentially with goitre, was published in 1894 at Milan and, in a 2nd edition, in 1895 at Trieste. Especially in the final chapter on therapeutics, Usiglio reveals himself as a fervent follower of Kocher.


Surgery for exophthalmic goitre in Australia, 1907.

Thomas Dunhill 1876-1957, Australian born surgeon (Melbourne 1907-1914 and London 1920-1941) performed partial thyroidectomy for exophthalmic goitre using Th. Kocher's method of local anaesthesia on seven consecutive severely toxic patients in 1907 with a successful outcome in all seven. A brief outline of the life and achievements of Thomas Dunhill is appended.


[Relations of Theodor Kocher with the "International Society of Surgery". His role as the 1st congress president].

With the aim of promoting progress in surgery through the friendly exchange of views and experience, the first International Society of Surgery was founded at Brussels in 1902, hereby helping to overcome the narrow boundaries of that times' nationalism. At its first congress, the "International Society of Surgery (ISS)", otherwise known by its French name "Société Internationale de Chirurgie (SIC)", numbered already 638 members, amongst them the most important surgeons from all over the world. Theodor Kocher was the president of the first congress, held at Brussels in 1905, and was also responsible for the choice of topics. His presidential address clearly reflected the high aims the Society set itself. Kocher's personal and professional authority, his surgical skill, which he liked so much to communicate to his colleagues, and his internationally minded thinking shaped the young society. He remained in the international committee of the ISS until his death in 1917.


[The Zurich lazarette in the wars of 1798/99. A manuscript by the hospital physician Johann Ludwig Meyer].

A manuscript, written about 1825, by the Zurich surgeon Johann Ludwig Meyer (1782-1852), is kept at the Institute and Museum of Medical History of the University of Zurich. The author describes his surgical observations in the makeshift military hospitals in Zurich during the French occupation of 1798 and the 2nd Coalition War in 1799. Together with Johannes Grimm, the then 16-year-old Meyer worked as an assistant to his father Konrad Meyer, the senior surgeon of Zurich, giving surgical treatment to wounded officers and soldiers in the French, Austrian and Russian military hospitals.


[Heavy metals in the mother-newborn infant biological system in the technology-related biogeochemical environment].

High levels of the lead, cadmium and zinc content in the mother and newborn blood, placenta, meconium and breast milk in the zinc production metallurgical works area were shown. Intoxication of newborns may be caused by breast feeding with milk containing heavy metals.


[Estimation of current maximum permissible levels of total specific activity of radionuclide mixture in the liquid forms of drugs and medicinal raw materials].

Algorithm of assessment of allowable levels of the total specific activity of radionuclides in liquid medicaments and medical raw materials was worked up. The key radionuclides were Strontium-90, Caesium-134 and -137.


[Hygienic evaluation of the physical activity programs for schoolchildren].

Theoretical prerequisites for a complex approach to making the hygienic standards of schoolchildren's motor regimes are discussed. Experimental evidence for the impact of the regimes on the development of schoolchildren is given.


[Evaluation of the neurobiological defense system of the body after exposure to complex environmental factors].

Effect of the air and water pollution complex on the rat neurobiological defense system (emotional reactivity, aggressiveness, forming and keeping of memory "traces", social and sexual options and so on) was investigated. Changes in behavioral reactions were noted. These changes correlated with the level of pollution.


[Epidemiology of abortion. A one-year prospective study].

This study evaluates the epidemiological characteristics of patients attended at the Hospital Central Militar (Service of Obstetrics), who had diagnosis of abortion. The study was prospective and was conducted through one year. There were 316 cases of abortion (12.4%) among 2,550 obstetrical patients. The most frequent type of abortion was the incomplete one (58.6%). Only 38 (12.0%) women had an septic abortion. From an epidemiological point of view, patients with abortion were young (mean age 26.45 +/- 6.49 years); married (87.4%); with mean parity of 2.20 +/- 2.16; 26.3% of them had their first pregnancy and 78.2% had their first abortion. Abortion were more frequent between 9 and 12 weeks of pregnancy. After the 10th week, the D&C had more complications than before. The conclusion from this study is that in this group of population, abortion is not an important problem of health.


[Correlation of creatine phosphokinase blood level with prenatal fetal heart rate as a prognostic factor in tissue hypoxia].

The presence of a high serum activity of the creatinine phosphokinase enzyme (CPK) could be the result of an hypoxic tissue event. The existence of an ominous fetal heart rate tracing is a reliable method which indicates the presence of an hypoxic state in variable degrees. Thirty-five pregnancies between 34 and 41 weeks of gestation were prospectively studied to correlate both, CPK activity and cardiotocography, with perinatal morbidity and mortality. All the patients had antepartum fetal heart rate testing and pregnancy was terminated by cesarean section within seven days to the last fetal heart tracing. As soon as the baby was born, we took an umbilical cord sample to measure CPK activity and a second sample was also taken at 36 hours of life. All the neonates had pediatric, neurologic, electrocardiographic and sonographic evaluation within their 48 hours of extrauterine life. Two groups were created: Group A included 14 neonates with normal cardiotocographic tracings (control group) and Group B had 21 infants with abnormal tracings (study group). We found an elevated serum CPK activity with statistic significance in the next three conditions: a) In the sample at 36 hours of life when compared to the cord sample in the control group, p less than 0.001; b) In the neonatal sample at 36 hours of age when compared to the cord sample in the study group, p less than 0.001; c) In the neonates of the study group compared to the neonates of the control group at 36 hours of extrauterine life, p less than 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)


[Laparoscopic findings during bilateral tubal obstruction].

During the period 1987 to 1990 at Hospital Luis Castelazo Ayala, IMSS, a prospective study was carried out. For this study 374 bilateral tubal obstructions (BTO) were included in interval of gestation by means of laparoscopy with silastic rings. The objective was to observe the status presented by the organs contained in the pelvic cavity of women considered healthy; so, as to present pathology, opportunely treat it. Main findings were: Pelvic adhesions 37 cases (9.8%), uterine myomatosis 33 cases (8.8%), tubal pathology 18 cases (4.8%), ovarian cysts 11 cases (2.9%), and endometriosis 9 cases (2.4%). Global pathology was 108 cases (28.8%). The use of laparoscopy in BTO allows to find non expected pathology in apparently healthy women and offers them the opportunity of receiving treatment immediately, avoiding thus to attribute to BTO symptomatology that could be derived from existing pathology.


[Kallmann syndrome: report of a pregnancy case and review of the literature].

We report the case of a pregnancy in a patient with Kallmann's syndrome. The patient responded adequately to gonadotropin releasing hormone (GnRH) stimulation test, and after the third cycle of stimulation the patient became refractory to the treatment. The patient did not respond to exogenous follicle stimulating hormone (FSH) or human menopausal gonadotropins (hMG) for ovulation induction. For these reasons, we used a short course of leuprolide acetate (LUPRON) followed by hMG, which resulted in ovulation and a subsequent pregnancy which was carried to term. We present an alternate approach to ovulation induction in patients with Kallmann's syndrome and a review of the literature.


[Nosocomial infection during puerperium].

Material and methods used to assess the epidemiology of puerperal infection (PI) in the National Institute of Perinatology (Mexico) between 1984 and 1990, are described. We observed that the general rate of PI ranged between 1.6 and 3.1; post-cesarean section posed a higher risk of infection than vaginal partum. Endometritis, associated with cesarean section, was the most frequent form of PI and gram positive flora was the most frequently found etiological agent. In general terms, we found that the rate of PI remained constant through the years of study, even when there was a significant increase in the practice of cesarean operations. Finally, we emphasize the need for the standardization of clinical norms used to assess the epidemiology of infection events. Standard norms will allow health-service institutions to compare results, observe tendencies, predict changes and take preventive actions.


[Radiotherapy of cancer of the endometrium. Analysis of 53 cases].

Fifty three patients with endometrial carcinoma received radiotherapy from 1986 to 1987, at the Hospital de Oncologia Centro Medico Nacional. Radiotherapy was given preoperatively in five patients, postoperatively in thirty nine patients, and radical in nine cases. Obesity, Hypertension and Diabetes were present in 60%. The patients have been in control from 3 to 44 months, with average of 18 months. Diagnosis was realized for genital bleeding 45/53 (85%), and increased uterine size 6/53 (11%). There were stage I 24/53 (45%), stage II 13/53 (24%) patients. Non classified eight cases, five of them were without tumoral activity at initial valoration, and three had tumor present. We analyzed stage treatment utilized, correlated with morbidity, tumoral response, free survival. We concluded that staging surgery is effective to chose the type of treatment.


[Tuberculosis in the pregnant woman].

The prevalence of tuberculosis (TB) in Mexico as in the rest of the developing countries is high and no reduction has been noted in the past years. This article describe the clinical features of the disease during the gestational period. We studied 15 cases of TB and pregnancy treated at the Infectious Diseases Department of the Instituto Nacional de Perinatología, from January 1990 to June 1991. At our Institute the incidence was 1.54 cases by 1000 obstetric patients discharged, with a lethality rate of 6.6 deaths by 100 infected patients. We treated 15 women, nine had pulmonary TB, three renal TB, two TB of the cervical lymph node and one cutaneous TB. In seven patients the diagnosis was made before pregnancy, other seven were diagnosed during gestation, and one during puerperium. All patients were treated with antituberculous drugs; the most frequent combination was isoniazid, ethambutol and rifampin. Twelve women had no complications, one died, another developed preeclampsia, and in one case the patient had a low birth weight neonate. No cases of congenital TB were identified and none of the neonates had abnormalities at birth.


[Changes in glucose metabolism during pregnancy: hospital experience].

The clinical heterogeneity of Diabetes Mellitus (DM) is also evident during the gestational period and thus, pregnancy could be complicated by a previously diagnosed DM or by diabetes that is first diagnosed during pregnancy (gestational diabetes or gestational alteration of the oral glucose tolerance test according with the degree of hyperglycemia). Independently of the stage at time of maternal diagnosis, the conceptus is at greater risk (probably since the time of conception) for abortion, genetic malformations, perinatal metabolic complications and death; these risks are apparently directly related with the time at diagnosis, duration and degree of metabolic alteration on the mother (mainly hyperglycemia) and the adaptive mechanisms on the product (hyperinsulinemia). Retrospectively, 412 pregnancies complicated with any type of carbohydrate metabolism alteration were studied in our service. The results demonstrated a high frequency of Gestational diabetes (42.2%) and of type II diabetes (35.9%); there was a good agreement with previous reports regarding the personal and family histories in the patients already known diabetic before pregnancy. The types of obstetric complications were similar to previous reports, but some of them with a greater frequency in our patients, namely hydramnios, toxemia, and urinary tract infection, and ketoacidosis with a minor frequency. We also observed an increased frequency of congenital malformations on the products. On the other hand, the metabolic complications of the newborn were similar to other reports with a slight predominance on the babies of known diabetic mothers prior to gestation.(ABSTRACT TRUNCATED AT 250 WORDS)


[Effect of primary ovarian insufficiency on the molecular heterogeneity of pituitary gonadotropins].

It is known that hypophysial gonadotropins circulate in different molecular forms which are provided with different biological activity and that the proportion of such varieties keep a relation with the concentrations of estrogens in circulation. In physiological menopause predominate the great molecular forms which have a lesser biological activity and when estrogens are administrated a change is produced to smaller molecular forms. The present study tries to determine if in cases of ovarian insufficiency of long duration as in precocious menopause and the syndrome of refractory ovary the chromatographic conduct of gonadotropin is similar. Six patients with precocious menopause, and three with the syndrome of refractory ovary, were studied, in order to make a comparison of chromatographic pattern of FSF and LH in serum, with the one found in women with normal ovarian function and with physiologic menopause. The chromatographic analysis was done by filtration in gel of the serum and the subsequent determination of LH and FSH by RIA of double antibody. It was found that the chromatographic pattern of patients with ovarian insufficiency of long duration, characteristically presents larger molecular forms, specially in the refractory ovary syndrome with a greater alteration in the profile of LH. With base on these results it is concluded that the duration of ovarian insufficiency determine the type of molecular form, predominant, of gonadotropins. Also it is suggested that for the syndrome of refractory ovary, the greater heterogenicity of gonadotropins may explain part of physiopathogeny.


[Mechanism of action of steroid hormones. I. Estrogens].

The steroid hormone are very versatile molecules: although they are related among them by their chemical structure, they have very diverse functions and including antagonic. Their action mechanism is not completely cleared. The estrogens participate in the regulation of practically all the reproductive and sexual events of the female, although the intracellular actions by which they take place are not well known and the proposed models do not adequately satisfy the questions. Currently it is accepted the existence of a cytoplasmic and/or nuclear receptor, without explaining satisfactorily how the hormones come to the nucleus. The endocrine events that are rapidly expressed (seconds) are due to a possible interaction with cellular membrane. The purpose of this review is to analyze and concilliate the reported data on the mechanism of action of estrogens.


[Ovarian cancer (experience with 186 patients)].

An experience with 186 ovarian carcinomas seen at the Oncology Service of The General Hospital of Mexico, between 1981 to 1990 is reported. Fifty patients, (26.9%) were in stage I; 2, (1.0%) were in stage II; 113 in stage III, (60.7%) and 21, (11.2%) were in stage IV. Epidemiological and clinical aspects are reviewed as well as results of treatment. In 137 cases, (73.6%) we have follow up and it was without evidence of disease between 12 and 60 months with a mean of 18 months in 32, (23.3%). This number includes 22 of 26 patients in stage I, (84.6%); 1 of 2 in stage II; 9 of 89, (10.1%) in stage III and 0 of 20 in stage IV. There were not significant differences in prognosis for stage I with the schemes of treatment employed: only surgery, postoperative radiotherapy and postoperative chemotherapy. Patients in stage III evolutioned without evidence of disease when the surgery left residual disease of 2 cms or less for unit: There were 1 of 59 patients treated only with surgery (1.6%); 3 of 15, (20%) treated with radiotherapy to whole abdomen plus over dosage to pelvis and 5 of 15, (33.3%) treated with two agents of chemotherapy. (cyclophosphamide plus adriamycin of cyclophosphamide plus cisplatin). In 3 of 11 stage III patients, (27.2%) with second look surgeries, we found macroscopic residual tumor.


[Premature membrane rupture: morbidity-mortality in pregnancies under 36 weeks].

The incidence of premature rupture of membranes in pregnancies of 36 weeks or less, in relation to the total births (3,796) is 1.3%; in relation to pregnancies less than 36 weeks (49 cases) 21.5%, and in pregnancies of less than 34 weeks (17 cases) of 0.47%. Infectious mortality was 2.2%. There was no maternal mortality. Cesarean section incidence was 43%. Neonatal infectious morbidity was present in 16.3%. Neonatal mortality on 4/30 cases was 13.0%; global mortality at two years was 5/30 cases, 17.0%.


[Nifedipine in gynecology and obstetrics].

Nifedipine has both vasodilator and relaxant actions in arterial smooth muscle and other tissues, like uterus. The goal of this review is describe the potential uses of this calcium antagonist in two clinical features in pregnancy. First, in premature labor, nifedipine is an appropriate alternative related with beta-mimetic or prostaglandin synthetase inhibitors agents, with good tocolytic properties and safe for both mother and baby. Second, in the treatment of hypertension in pregnancy or preeclampsia, where, according literature, nifedipine would be a valuable therapeutic gun. It is exposed the nifedipine advantages and disadvantages in pregnancy found by the authors from the reports reviewed here. It is analyzed the ethics of nifedipine use in pregnancy. Moreover, it is pointed out the nifedipine clinical usefulness in the treatment of primary dysmenorrhea and it is analyzed some nifedipine hemodynamic aspects on uterine blood flow.


[Report of a case of deferred abortion in cervical pregnancy].

A patient with cervical pregnancy by clinical and ultrasonographic criteria underwent cervical and uterine curettage without complications. The histological study showed hyalinized corial villous with inflammatory infiltration and the material from uterine cavity showed decidual changes. The spontaneous and previous embryo death induce a scanty bleeding following curettage and thus, corroborate in a natural way the efficacy of any substance used to desvitalize the pregnancy and in consequence, diminish the vascular blood flow before evacuation in order to get a good result following conservative management.


[Quantification of deoxyribonucleic acid and collagen as biological markers in uterine leiomyoma].

The uterine leiomyomas (UL) are tumors compound by smooth muscle connective tissue. Growth depends mainly of two basic mechanisms: hypertrophy of myometrial cells and connective tissue deposit. The quantification of the basic elements of the tumor permits to try its application as biochemical markers of disease, auxiliary diagnostic criteria, or in medical therapeutical monitorization, alternative that lately has become very important. In the present study 33 patients are included, in two groups. Group I women with uterine leiomyomatosis (n = 17) and Group II (n = 16) without UL. A prospective, double blind, transversal of cases and control study. Connective tissue concentration was evaluated based on collagen determination. The evaluation of muscular tissue was done by desoxyribonucleic acid (DNA) measurement. The results showed a significant increase in connective tissue concentration (until 500%) and a significant diminution of cellularity (DNA) in women with UL, as compared with control group. The main biochemical and clinical implications of these findings, are commented upon.


Human plasma lipid peroxide levels show a strong transient increase after successful revascularization operations.

This study was performed to evaluate the hypothesis that oxygen radicals/lipid peroxidation are involved in reperfusion injury in humans. The study included 37 patients, who underwent surgical revascularization operations for kidney transplantation (9 subjects) or limb salvage (28 subjects). Peripheral venous blood samples were taken 30 min before starting reperfusion (baseline) and 1, 2, 3, 4, and occasionally 6 to 18 h after revascularization. The amount of plasma malonaldehyde formed in the reaction with thiobarbituric acid (MDA-TBA) was determined by high-performance liquid chromatography (HPLC). The baseline MDA-TBA values of the patients were very close to the value determined for 20 age-matched healthy subjects (i.e. mean +/- SD 0.689 +/- 0.294 nmol/mL plasma [range 0.2 to 1.37] vs. 0.700 +/- 0.209 nmol/mL plasma [range 0.385 to 1.29]). All patients responded to successful revascularization with significant increase of the plasma MDA-TBA within about 1 h after onset of reperfusion. Thereafter the values decreased nearly to the preoperative state. The mean increase of MDA-TBA was 107% in kidney transplantation and 54% in limb revascularization. In a few patients with severe arteriosclerosis, revascularization was not optimal and no increase in the MDA-TBA value occurred. The results of this study indicate that therapeutic intervention to prevent lipid-peroxidation-mediated reperfusion injury is confined to a rather narrow time window and must be undertaken either prior to or immediately after revascularization.


Selection and analysis of superoxide dismutase mutants of Neurospora.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.


Formation of hydrogen peroxide by lens proteins: protein-derived hydrogen peroxide as a potential mechanism of oxidative insult to the lens.

The exposure of dialyzed preparations of lens crystallins to copper (II) ions causes a decrease in protein surface thiol and the production of hydrogen peroxide (H2O2). H2O2 production by gamma and beta crystallin subfractions (which contain the greatest level of thiol) is the predominant source of this H2O2. Protein surface thiols are probable sources of H2O2 formation since N-ethyl maleimide treatment of lens proteins and zinc ions inhibit H2O2 production. These data are consistent with a hypothesis that transition metal-catalyzed oxidation of protein contributes to cataractogenic lens protein oxidations.


Brown fat thermogenesis and exercise: two examples of physiological oxidative stress?

Both brown fat tissue (BAT) and skeletal muscle experience large increases of oxygen consumption and oxygen radical generation during activation. This, together with the relatively low activities of antioxidant enzymes in these two tissues and the high lipid content and free fatty acid liberation of BAT, can produce a physiological oxidative stress. Increases of in vivo or in vitro (BAT) lipid peroxidation have been described in these tissues after activation. They react to this oxidative stress in an adaptive way after chronic stimulation. Cold acclimation increases antioxidant enzymes, ascorbate, and especially reduced glutathione (GSH) in BAT. There is controversy about the variations of antioxidants in skeletal muscle after acute exercise. Nevertheless, exercise training seems to increase muscle antioxidant enzymes and GSH. Many reports show that vitamin E levels decrease in the muscle and increase in plasma during exercise. Studies of vitamin E deficiency and supplementation strongly suggest that this vitamin is of protective value during exercise.


The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.


Biochemical and toxicological properties of the oxidation products of catecholamines.

The normal catabolism of catecholamines proceeds through enzymatic pathways (monoaminooxidase, catechol-o-methyltranserase, and phenolsulphotransferase). In addition, nonenzymatic oxidative pathways might take place since catechols are readily oxidized. In this review article, the pathways of formation of the oxidation products of catecholamines and their reactions are described. The interactions of these products with different biological systems and their toxicity are examined. Among the reactions known to occur is that with sulfhydryls, which results in either a covalently linked adduct or disulfide production. Another interesting pathway to toxicity involves the oxidation of these catecholamine products by oxygen, with the formation of damaging oxygen-derived species. The action of the oxidation products of catecholamines is outlined, with special attention to the nervous and cardiac systems.