Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid. A cDNA clone coding for a sea urchin embryonic protein was isolated from a prehatching blastula lambda gt11 library. The predicted translation product is a secreted 64 x 10(3) Mr enzyme designated as BP10. The protein contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP10 protease is constructed with the same domains as the human bone morphogenetic protein BMP-1, a protease described as a factor involved in bone formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP10 gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP10 transcripts is limited to a short period at the blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP10 transcripts were only detected in a limited area of the blastula, showing that the transcription of the BP10 gene is also spatially controlled. Antibodies directed against a fusion protein were used to detect the BP10 protein in embryonic extracts. The protein is first detected in early blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP10 protein during its synthesis and secretion was analysed by immunostaining blastula-stage embryos. The intracellular localization of the BP10 staining varies with time. The protein is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70% of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP10 protein and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP10 in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP10 in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.


Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens. The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.


ming is expressed in neuroblast sublineages and regulates gene expression in the Drosophila central nervous system. Cell diversity in the Drosophila central nervous system (CNS) is primarily generated by the invariant lineage of neural precursors called neuroblasts. We used an enhancer trap screen to identify the ming gene, which is transiently expressed in a subset of neuroblasts at reproducible points in their cell lineage (i.e. in neuroblast 'sublineages'), suggesting that neuroblast identity can be altered during its cell lineage. ming encodes a predicted zinc finger protein and loss of ming function results in precise alterations in CNS gene expression, defects in axonogenesis and embryonic lethality. We propose that ming controls cell fate within neuroblast cell lineages.


Splicing with inverted order of exons occurs proximal to large introns. Following our studies which showed that the alpha and beta exons of the chicken c-ets-1 gene are not conserved in the human homologue, we succeeded in identifying a novel human c-ets-1 transcript in which the normal order of exons is scrambled. By PCR and RNase protection assays, we demonstrated that while the order of exons is different from that in genomic DNA, splicing of these exons in aberrant order occurs in pairs and at the same conserved consensus splice sites used in the normally spliced transcript. The scrambled transcript is non-polyadenylated and is expressed at much lower levels than the normal transcript. It is not the consequence of genomic rearrangement at the ets-1 locus nor is it due to the transcription of any ets-1 pseudogene. These results confirm previous observations of scrambled splicing.


Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo. The initiation of protein synthesis on foot-and-mouth disease virus RNA occurs at two sites separated by 84 nucleotides. Immediately upstream from the first of these sites is the internal ribosome entry site (IRES), which directs the translation of this RNA to be cap-independent. The utilization of these two initiation sites has been examined using artificial fusion genes in vivo under a variety of conditions. Additional in-frame AUG codons have been introduced between these two authentic start sites to determine the mechanism by which ribosomes recognize the second start site. The results indicate that following internal entry of ribosomes on the 5' side of the first initiation codon, many fail to initiate protein synthesis at this position and scan along the RNA to the second initiation site. In the presence or absence of the IRES both initiation sites are efficiently used but the utilization of the two sites is slightly biased towards the second initiation site by the IRES. Furthermore, in the presence of the IRES, protein synthesis initiates at both sites independently of the activity of the cap-binding complex.


Horizontal transfer of a phosphatase gene as evidence for mosaic structure of the Salmonella genome. The genomes of Escherichia coli and Salmonella typhimurium are similar with respect to base composition, chromosome size, and the order, orientation and spacing of genes, but differ with respect to some 29 'loops', regions unique to one species. To evaluate the genetic basis for the structure and organization of the enteric bacterial genomes, we examined the gene encoding a non-specific acid phosphatase (phoN) which maps to a loop at 96 min on the S.typhimurium chromosome. We detected atypical base composition, codon usage pattern and trinucleotide frequencies. The 1.4 kb region containing phoN had an overall base composition of 43% G+C, while the G+C content at the third positions of codons in the phoN reading frame is only 39%, much lower than the Salmonella chromosome which averages 52%. Non-specific acid phosphatase activity, assayed in 14 Gram-negative species, was detected only in Morganella morganii and Providencia stuartii, organisms with low genomic G+C contents. Upstream of the phoN gene in Salmonella is a sequence with high similarity to the oriT region of incFII plasmids, suggesting that the phoN gene, and perhaps the entire loop structure, was acquired by lateral transmission in a plasmid-mediated event.


Identification of NF-jun, a novel inducible transcription factor that regulates c-jun gene transcription. In this study we report the identification of a novel transcription factor, termed Nuclear Factor-jun (NF-jun). This factor contributes to inducible transcription of the c-jun gene in human myeloid leukemia cells. NF-jun was, however, undetectable in nuclear proteins from human monocytes, granulocytes, resting T lymphocytes and lung fibroblasts. NF-jun shares several features with the well characterized NF-kappa B in that binding activity can be generated in cytosolic extracts by treatment with dissociating agents. In addition, binding of NF-jun to its recognition site is enhanced by treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, tumor necrosis factor alpha or the protein synthesis inhibitor cycloheximide (CHX). However, as revealed by competition assays and electrophoretic mobility shift assays, purified NF-kappa B fails to bind to the c-jun fragment which contains the NF-jun site, and this fragment fails to compete with NF-kappa B for binding. UV crosslinking showed that NF-jun contains a 55 and a 125 kDa protein species. These findings demonstrate that the c-jun gene can be regulated by a transcription factor distinct from AP-1. Our findings also indicate that while NF-jun has several features in common with the NF-kappa B binding protein including its subcellular localization and its ability to translocate from the cytoplasm to the nucleus, this factor recognizes a unique DNA sequence. Moreover, the activity of this protein is differentially regulated in various cell types. NF-jun might function as a signal transducing molecule in order to mediate rapid induction of the early response gene c-jun in a cell type- and stimulus-specific manner.


Prolactin message in brain and pituitary of adult male rats is identical: PCR cloning and sequencing of hypothalamic prolactin cDNA from intact and hypophysectomized adult male rats. Prolactin (PRL), or a PRL-like molecule has been identified in the central nervous system and other tissues by numerous investigators. The previous finding of PRL in brain persisting for weeks following hypophysectomy led us, and others, to conclude the brain and central nervous system PRL is synthesized locally. Also, our previous results showing PRL mRNA in hypothalamic and extra-hypothalamic brain regions using reverse transcription-polymerase chain reaction (RT-PCR) techniques, along with this report that the sequence of the PRL message in the brain is identical to that found in the anterior pituitary solidifies our, and others, hypothesis that PRL is synthesized in many locations other than the traditional one (anterior pituitary). The actual sequencing of hypothalamic PRL cDNA produced from RT-PCR of mRNA from intact or hypophysectomized rats demonstrates unequivocally that brain PRL mRNA is identical to anterior pituitary prolactin mRNA.


The beta-tubulin genes of Paramecium are interrupted by two 27 bp introns. In the ciliate Paramecium tetraurelia, the analysis of the tubulin gene family has revealed the existence of four alpha and three beta genes. We show here that the coding sequence of the first beta-tubulin gene to be cloned and sequenced is interrupted by two short non-coding sequences of 27 bp each, which present at their extremities the pairs GT/AG, characteristic of eukaryotic pre-mRNA introns, and the internal pentanucleotide TTAAT, consensual in Tetrahymena introns. We demonstrate by PCR experiments that the three macronuclear beta-tubulin genes contain these sequences in similar positions, thereby ruling out the possibility that these sequences are ciliate IES present in the micronucleus and eliminated in the transcriptionally active macronucleus. S1 mapping analysis and mRNA sequencing show that the sequences are absent from the beta-tubulin transcripts. These sequences are the first introns described in protein encoding genes in P. tetraurelia and the shortest known introns altogether.


cDNA clones for mouse parotid proline-rich proteins. mRNA regulation by isoprenaline and the nucleotide sequence of proline-rich protein cDNA MP5. cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline-rich protein (PRP) oligonucleotide. They were divided into two cross-hybridisation groups, A and B, which were shown by hybrid-selected translations to encode acidic PRP and basic PRP, respectively. The A-type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B-type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B-type clones contained a novel PRP cDNA (MP5) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260-residue-long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGPPPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3'-untranslated region to rat salivary glutamate/glutamine-rich protein (GRP) cDNA. Two MP5 clones contained a 273-bp intron-like insertion in the 3' untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts. Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B-type PRP mRNA was present in normal parotid glands. RNA dot-blots probed with PRP-gene-specific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.


An additional exon in the human vinculin gene specifically encodes meta-vinculin-specific difference peptide. Cross-species comparison reveals variable and conserved motifs in the meta-vinculin insert. We have analyzed the structure, origin and expression of the high-molecular-mass muscle-specific variant of vinculin, called meta-vinculin. The meta-vinculin-specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross-species identity in the C-terminal half of the insert. Human and porcine meta-vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta-vinculin showed 22 exchanges in the same region compared to human meta-vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs. Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease-mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204-nucleotide insert that precisely encoded the meta-vinculin-specific peptide. Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta-vinculin-specific insert is present as a discrete exon. Thus, meta-vinculin and vinculin mRNA are generated by alternative splicing.


Type XIV collagen is a variant of undulin. We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.


Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the marmoset and rabbit genes. In mammals, the subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT) is species dependent, with the proportion of AGT targeted to mitochondria varying between 0% and greater than 90%, the remainder being located in the peroxisome. In order to extend our studies on the molecular evolution of intracellular targeting of AGT, we have investigated the organization and expression of the AGT genes of rabbit, which has all of its AGT located in the peroxisome, and marmoset, which has approximately 50% of its AGT located in the peroxisome and 50% in the mitochondrion. Southern-blot analysis indicates that, in both of these species, AGT is encoded by a single-copy gene, as has previously been shown for human (all AGT in the peroxisome) and rat (50% AGT in the peroxisome and 50% in the mitochondrion). Comparison of the cDNA sequences encoding marmoset, rabbit, human and rat AGT, combined with transcript mapping and in vitro mitochondrial protein-import analysis, has provided a molecular explanation for the differential targeting of AGT in these species. As in the rat, marmoset AGT is synthesized in two forms, via the use of alternative transcription and translation-initiation sites. These two forms of AGT differ only in the presence or absence of a 22-amino-acid amino-terminal peptide, which acts as a cleavable mitochondrial-targeting sequence, directing the longer form of AGT to mitochondria. The shorter form of AGT, lacking the mitochondrial-targeting sequence, is presumed to be localized in the peroxisomes. In humans and rabbits, similar but distinct evolutionary mutational events within the AGT gene have resulted in exclusion of the region encoding the mitochondrial-targeting sequence from the open reading frame, explaining the exclusive peroxisomal localization of AGT in these species. We discuss the impact of these results on our understanding of both the evolution of species dependence of AGT subcellular distribution and the recent identification of amino acid changes in human AGT which result in mistargeting of this protein to mitochondria.


The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough). The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.


Molecular analysis of the V kappa III-J kappa junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition. Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RF kappa variable regions from a patient with RA were encoded by V kappa III genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen-driven maturation. Because an important role of the light chain third complementarity-determining region (CDR3) in anti-IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged V kappa gens. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between V kappa and J kappa. The sequence analysis of our patients' germ-line Vg and J kappa 4 genes segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ-line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF kappa chains we suggest that "pathogenic" RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of V kappa and J kappa seem exceptional in expressed human kappa chains and because the resulting amino acid residue is a proline in most cases, we think that RF kappa chain CDR3 is under a very strong selective pressure during RA.


Immunoglobulin VH-T cell receptor C alpha fusion mRNA resulting from chromosome inversion include the T cell-associated 5' exon ET. A human T cell lymphoma has been described in which an inversion of chromosome 14 results in fusion of an immunoglobulin heavy chain VH with a T cell receptor J alpha segment, potentially resulting in a chimeric protein with immunoglobulin VH region recognition plus T cell receptor effector functions. Examination of the mRNA species expressed from the IgT gene in this lymphoma shows a variety of forms but all IgT mRNA include the T cell-specific exon, ET, previously located in the distal part of the VH locus. In such mRNA species, the normal leader exon of the Ig VH segment, which encodes most of the hydrophobic signal peptide, is replaced by the short ET exon encoding mainly non-hydrophobic residues. Two forms of this mRNA exist which lack the Ig VH leader sequence and thus potentially yield non-membrane proteins in the T cell lymphoma.


The lacrimal gland synthesizes retinol-binding protein. The rabbit lacrimal gland secretes retinol bound to a 20-21 kDa protein. To test the hypothesis that this protein might be retinol-binding protein (RBP) we probed lacrimal gland for RBP mRNA and lacrimal gland fluid for RBP. A rabbit RBP cDNA clone was used to probe rabbit and rat lacrimal gland RNA using RNase protection analysis. The lacrimal gland contains RBP mRNA at a level 0.1 to 0.03% of that observed in the liver. This RBP mRNA was identical to that observed in the liver based on RNase protection analysis, Northern blot analysis and primer extension analysis. The RBP mRNA levels in the lacrimal gland were not altered by the retinol status of the rabbits. We analysed lacrimal gland fluid for RBP by immunoblotting using a monoclonal antibody that recognizes rat, human and rabbit RBP. A single protein band from the rabbit lacrimal fluid bound the antibody, and this protein comigrated with human RBP which also bound the antibody. We conclude that the lacrimal gland contains RBP mRNA and that the lacrimal gland synthesizes and secretes RBP into the lacrimal gland fluid. This is the first demonstration that an extrahepatic tissue containing RBP mRNA synthesizes and secretes the protein in vivo.


Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. Malonyl transacylase has been overexpressed 155-fold compared to a wild-type strain. Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of cis-vaccenate are incorporated into the membrane phospholipids.


Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus. A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.


Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin. The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.


Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast. The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.


Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase. A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (G1DH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) mutants. Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da. The deduced amino acid composition and sequence of G1DH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent G1DH from B. stearothermophilus RS93. The enzyme has limited homology to the iron-activated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum.


A second gene (qutH) within the Aspergillus nidulans-quinic-acid utilisation gene cluster encodes a protein with a putative zinc-cluster motif. A sequence of 3299 nt, contiguous with the previously sequenced quinate permease-encoding (qutD) gene and encompassing the dehydroshikimate dehydratase-encoding (qutC) gene, has been determined. Northern-blot analysis detected (i) a quinate-inducible mRNA of the expected size for the qutC gene, and (ii) a quinate-inducible mRNA of 1.45 kb divergently transcribed away from qutC towards qutD. Computer-aided sequence analysis identified an ORF of 1047 nt corresponding to the qutC gene encoding dehydroshikimate dehydratase. In addition, a genetically uncharacterized 1188-nt gene, designated qutH and containing a putative intron of 61 nt, was identified between qutC and qutD. The inferred protein sequence encoded by qutH contains a putative 'zinc cluster' motif and has a low (16%) but significant similarity with the DNA-directed DNA polymerase of hepatitis B virus. The results are interpreted as being consistent with the view that the qutH gene encodes a DNA-binding protein, possibly involved in the regulation of genes essential for the utilisation of protocatechuic acid.


Rainbow trout p53: cDNA cloning and biochemical characterization. We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.


Cloning and sequencing of genes encoding the TthHB8I restriction and modification enzymes: comparison with the isoschizomeric TaqI enzymes. Genes encoding the TthHB8I restriction and modification (R-M) system from Thermus thermophilus HB8 (recognition sequence T decreases CGA) were cloned in Escherichia coli. The genes have the same transcriptional orientation, with the last 13 codons of the methyltransferase (MTase) overlapping the first 13 codons of the endonuclease (ENase). Nucleotide sequence analysis of the TthHB8I ENase revealed a single chain of 263 amino acid (aa) residues that share a 77% identity with the corrected isoschizomeric TaqI ENase. Likewise, the Tth MTase (428 aa) shares a 79% identity with the corrected sequence of the TaqI MTase. This high degree of aa conservation suggests a common origin between the Taq and Tth R-M systems. However, codon usage and G+C content for the R-M genes differed markedly from that of other cloned Thermus genes. This suggests that these R-M genes were only recently introduced into the genus Thermus.


Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli. A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins. The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein. Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation. This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.


Characterization of the gene encoding the most abundant in vitro translation product from virus-infected Chlorella-like algae. The gene (33kDa) encoding a 33-kDa peptide from Chlorella virus, PBCV-1, was cloned and sequenced. This gene encodes the most abundant in vitro translation product synthesized from viral mRNAs isolated beginning at 20 min post-infection. The message persisted throughout the remainder of the viral life cycle. An open reading frame (ORF) of 717 bp, which encodes a polypeptide of 238 amino acids with a predicted M(r) or 26,613, was found on a 2752-bp cloned fragment from PBCV-1 HindIII restriction fragment 9. Transcriptional analysis of this ORF indicated that it was expressed both early and late, and as the viral life cycle progressed, the mRNA increased in size and abundance. Three other ORFs were also found; the largest of which (741 bp) hybridized to a low-abundance transcript which would encode a polypeptide with a predicted M(r) of 27,854.


Sequence analysis of an insertion element, IS1131, isolated from the nopaline-type Ti plasmid of Agrobacterium tumefaciens. Transferred DNA (T-DNA) from several nopaline-type Ti plasmids of Agrobacterium tumefaciens was previously shown to be variable in size due to the insertion of extra DNA segments. We found an insertion sequence (named IS1131) in the T-DNA of the strain PO22 and determined the nucleotide (nt) sequence. IS1131 is 2773 bp long, contains four open reading frames, and is flanked by 12-bp perfect inverted repeats (IR). An 8-bp direct repeat was found immediately outside the IR, and represents a target site of integration. Although the IS1131 nt sequence showed a limited degree of similarity to those of the previously reported IS elements of A. tumefaciens and to the central region of T-DNA, the terminal IR of IS1131 were highly homologous (up to 83%) to those of IS66 and IS866, suggesting that these three IS elements are related to each other. A number of IS1131-related copies were found in several pathogenic, as well as nonpathogenic, nopaline-type strains from different plant sources, and were distributed on both chromosomes and plasmids.


Disruption analysis of metallothionein-encoding genes in Candida glabrata. Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-IIa, an amplified locus, and MT-IIb, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CuSO4 required to inhibit the growth by 50% (IC50) of a C. glabrata strain harboring an amplified MT-IIa locus and a single-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The IC50 decreased to approx. 1 mM when the amplified MT-IIa locus was deleted. The disruption of the MT-IIb gene decreased the IC50 further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-II genes was attributable to MT-I; no evidence was found for the production of (gamma EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-IIa revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-IIb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IIa gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.


Isolation, characterization and chromosomal localization of the human GADD153 gene. We report the isolation and characterization of the growth arrest and DNA-damage-inducible gene, GADD153, from human cells and show that it is localized in the region 12q13.1-q13.2 on chromosome 12. Comparison of the human gene with the previously described hamster gene revealed a high level of conservation in both the structural and regulatory regions of the genes. Each is composed of four exons with intron/exon junctions maintained at the identical positions. The human Gadd153 protein shares 91% identity with the hamster protein in amino acid sequence, and 78% identity in nucleotide sequence. A 900-bp fragment of 5' flanking sequence from the human gene, when linked to the bacterial cat reporter gene, was found to exhibit promoter activity in HeLa cells which could be further activated by treatment with the DNA alkylating agent, methyl methanesulfonate. Sequence analysis indicated that the human promoter region is relatively G+C-rich and contains putative binding sites for multiple transcription factors, including recognition sites for TATA- and CAAT-binding proteins, six Sp1-binding sites, an activator protein-1 binding site, an E-26-specific sequence-binding protein-1 DNA-binding site, and four interleukin-6 response elements. Many of these sites are also present in an identical position in the hamster gene suggesting they may play an important role in regulating GADD153 expression.


Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library. A rat brain cDNA library was screened with probes constructed from portions of the cDNA (OBCAM) encoding the opioid-binding cell adhesion molecule (OBCAM). Three clones of interest were isolated and sequenced. The largest clone, DUZ1, had a putative open reading frame (ORF) essentially identical to OBCAM in its C-terminal 318 amino acids (aa), but differing in its N-terminal aa (20 vs. 27 in OBCAM), and in all of its 5'-noncoding regions. The other clones, SG8 and SG13, had a putative ORF essentially identical to that of OBCAM, but differed from each other in a portion of their 5'-noncoding region. This study suggests that there is a family of OBCAM-like genes.


Cloning and expression of the Acanthamoeba castellanii gene encoding transcription factor TFIID. We have cloned and characterized the cDNA encoding transcription factor TFIID from the eukaryote, Acanthamoeba castellanii. The gene occurs as a single species, encodes one mRNA and, presumably, a single protein. A. castellanii TFIID contains two recognizable domains, a nonconserved N-terminal domain and a highly conserved C-terminal domain. Similarities between the amino acid (aa) sequences of TFIID from several organisms are also found within the N-terminal 78 aa, suggesting a potential role in TFIID function. Full-length or truncated A. castellanii TFIID produced in Escherichia coli binds to a TATA box and is able to activate transcription in a TFIID-depleted HeLa cell extract, but the C-terminal 180-aa domain was found to be less efficient in these reactions.


Nonidentity between plasmid and chromosomal copies of ISS1-like sequences in Lactococcus lactis subsp. lactis CNRZ270 and their possible role in chromosomal integration of plasmid genes. The nucleotide sequence of an insertion sequence (IS) observed during mating experiments using the lactose-protease plasmid, pUCL22, of Lactococcus (Lc.) lactis subsp. lactis CNRZ270, was found to be similar to that of ISS1 from Lc. lactis subsp. lactis ML3. The IS was named ISS1RS. The chromosome of this strain contains several copies of ISS1-like IS as assessed by hybridization. One of these copies was cloned and named ISS1CH. Its sequence differs from that of the plasmid-borne copy, and appears to be more closely related to ISS1N from Lc. lactis subsp. cremoris SK11. This suggests independent introduction of both ISS1 elements. Moreover, the observation of plasmid genes integrated in the CNRZ270 chromosome near ISS1CH suggests that their presence is the result of integration by a Campbell mechanism using both IS homologies. ISS1-like sequences were also found on plasmids of numerous Lc. lactis strains, as well as one out of seven Lactobacillus (Lb.) casei and one out of three Lb. plantarum strains examined.


Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids. A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.


Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase. The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.


Sequence of rat lipoprotein lipase-encoding cDNA. A rat lipoprotein lipase (LPL)-encoding cDNA (LPL) has been entirely sequenced and compared to the sequences of all the LPL cDNAs reported in other species. As expected, high homology was found between the coding exons. The putative catalytic triad, Ser132, Asp156, His241, according to human numbering, is conserved in rat. As is the case in mouse, an Asn444 present in human LPL is also missing. The major divergences between human, mouse and rat LPLs were observed in the untranslated exon 10, where (i) the rat cDNA exhibits a 157-bp insertion and an 81-bp deletion relative to human; (ii) neither the B1 repeat nor the homopurine stretch reported in mouse can be recognized, and (iii) the rat cDNA displays several A+T-rich stretches.


Characterization of a new member of the sea urchin Paracentrotus lividus hsp70 gene family and its expression. We have sequenced a second gene of the hsp70 family derived from a genomic clone of the sea urchin, Paracentrotus lividus. The structure of this gene, named hsp70IV gene, is interrupted by one intron and differs from the previously analyzed sea urchin hsp70II gene, which contains several introns. Two open reading frames of hsp70IV gene encode a predicted protein of 639 amino acids with an M(r) of 69,672. The 5' flanking region of the gene contains a putative TATA element, three heat-shock elements made up of some arrays of the 5-bp units, NGAAN and NTTCN (N = A,C,G or T), a canonic consensus sequence for binding of the regulatory activating transcription factor (ATF), and a purine box. The 3' flanking region contains four putative polyadenylation sites located at different sites downstream from the stop codon. Using Northern blot hybridization analysis, carried out using a probe corresponding to a 3' noncoding fragment (UTR) peculiar to hsp70IV gene, we found that this gene is transcribed only under heat shock (Hs) and that the transcript can be recovered from the polysomal pellet. The hsp70IV gene may be classified as a Hs gene 70 although it contains one intron.


Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae. A gene (FDH1) of Candida maltosa which confers resistance to formaldehyde in Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The gene has a single intron which possesses the highly conserved splicing signals found in S. cerevisiae introns. We demonstrated that processing of the pre-mRNA of the cloned gene occurred identically in both S. cerevisiae and C. maltosa. The predicted amino acid sequence from the cloned gene showed 65.5% identity to human alcohol dehydrogenase (ADH) class III and 23.9% identity to S. cerevisiae ADH1. The most probable mechanism of resistance to formaldehyde is thought to be the glutathione-dependent oxidation of formaldehyde which is characteristic for ADH class III. The cloned FDH1 gene was successfully employed as a dominant selectable marker in the transformation of S. cerevisiae.


Regulation of alpha-amylase-encoding gene expression in germinating seeds and cultured cells of rice. Four alpha-amylase-encoding cDNA (alpha Amy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA3)-treated rice aleurone layers. Nucleotide sequence analysis indicates that the four cDNAs were derived from different alpha Amy genes. Expression of the individual alpha Amy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes. In germinating seeds, expression of the alpha Amy genes is positively regulated by GA3 in a temporally coordinated but quantitatively distinct manner. In cultured suspension cells, in contrast, expression of the alpha Amy genes is negatively and differentially regulated by sugars present in the medium. In addition, one strong and one weak carbohydrate-starvation-responsive alpha Amy genes have been identified. Interactions between the promoter region (HS501) of a rice alpha Amy gene and GA3-inducible DNA binding proteins in rice aleurone cells were also studied. A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501. One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA3-response element. The other fragment is located between nt -92 to -130 that contains a putative enhancer sequence. The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.


A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences. Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.


A variant limb deformity transcript expressed in the embryonic mouse limb defines a novel formin. The formins constitute a set of protein isoforms encoded by the alternatively spliced transcripts arising from the limb deformity (ld) locus of the mouse. Mutations in this locus disrupt formation of the anteroposterior axis of the embryonic limb. Although ld transcripts are widely expressed during embryogenesis, we have identified a novel transcript that is expressed in the mesenchyme and apical ectodermal ridge of the developing limb. This pattern of expression coincides with the earliest morphological defects observed in ld mutant limb buds. Moreover, the formin encoded by this transcript bears a highly acidic amino terminus, as distinguished from the basic amino terminus encoded by other ld transcripts suggesting that it may have a distinct biochemical function.


The embryonically active gene, unkempt, of Drosophila encodes a Cys3His finger protein. The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys3His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development.


Meiotically induced rec7 and rec8 genes of Schizosaccharomyces pombe. The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.


[Structural and functional analysis of the promoter region of the mts1 gene]. The mts1 gene is specifically expressed in metastatic tumors but not in non-metastatic counterparts. The gene was cloned from both mouse and human sources. The 5'-flanking regions of the mts1 gene from both sources were sequenced, revealing a 135 base pair region of high homology in the vicinity of the TATA box. The 5' region of the mts1 gene was also observed to have high degree of homology with some known promoter and enhancer sequences. The results of our transient transfection assays, in conjunction with those obtained from in vivo and in vitro footprinting of the promoter region, show no evidence of cis-control elements important for transcriptional regulation of mts1 gene. mts1 was found to be hypermethylated in CSML-0 cells and in the tissue types that do not express the gene or do that only at low levels. The possible role of methylation in progression of the nonmetastatic CSML-0 adenosarcoma cell line towards the metastatic CSML-100 adenosarcoma cell line will be discussed.


Construction of a GT polymorphism map of human 9q. To construct a framework map of human chromosome 9 consisting of highly informative markers, we identified 36 cosmid clones from chromosome 9 that contained long GT repeat sequences. The cosmids were found to cluster on the long arm of the chromosome, particularly in the q32-34 region. Thirteen highly informative polymorphisms from 9q were identified, with median observed heterozygosity 0.75 and median calculated heterozygosity based upon allele frequencies of 0.75. These new GT repeat polymorphisms (D9S56, D9S58-67), as well as anchor GT polymorphisms for D9S15 (MCT112, 9q13), and ABL and ASS (both 9q34.1) were utilized to construct a linkage map of human 9q by the typing of the Venezuelan Reference Pedigree. Care was taken to avoid errors, including analysis of the data with CHROMLOOK and verification of all double crossover events detected within a 30 cM interval by repetition of the marker analysis. The map was generated using the MAPMAKER program. All positions in the resulting map are favored by odds of greater than 10(4):1. The map has a sex-averaged length of 90 cM (Kosambi function) with a single maximum intermarker recombination fraction of 26%. All other intermarker recombination fractions are less than 15%. As D9S15 is known to be closely linked to markers on proximal 9p, and ASS/ABL are in band 34.1, this set of GT polymorphisms spans the length of 9q and provides a useful panel for linkage analysis of disease genes to this region. The marker order was confirmed by in situ hybridization of the cosmid clones to metaphase spreads of normal human chromosomes, which indicated an excess of recombination in the telomeric region in comparison to centromeric 9q, in agreement with previous chiasmata distribution observations. Two spontaneous new mutations for these GT repeat markers were identified, giving an overall observed spontaneous mutation rate of 0.00045 per locus per gamete. Direct observation of new mutations has not been previously reported for dinucleotide polymorphisms, but the observed rate is consistent with frequencies observed for other VNTR polymorphisms.


Concerted evolution of the mouse Tcp-10 gene family: implications for the functional basis of t haplotype transmission ratio distortion. The mouse Tcr locus is defined by its central role in the transmission ratio distortion phenotype characteristic of t haplotypes. A molecular candidate for Tcr has been identified in the form of a gene--Tcp-10b--expressed during spermatogenesis. Tcp-10b is one member of a multigene family present in two to four copies on different homologs of chromosome 17. The coding regions of the Tcp-10 genes present within two inbred strains were compared with those of the tw5 haplotype. The various gene family members are highly conserved relative to each other with a minimum nucleotide identity of 98.6% in all pairwise comparisons. Maximal parsimony analysis indicates that the Tcp-10 gene family has evolved in a concerted manner with the obliteration of nearly all individual gene-specific characteristics. As a consequence, the candidate for the full-length mutant Tcr gene product is distinguished by only a single, highly conservative, amino acid change. The data are consistent with the hypothesis that the effector of mutant Tcr activity is a second, alternatively spliced product that is expressed in a haploid- and allele-specific manner.


Human choline acetyltransferase (CHAT): partial gene sequence and potential control regions. Choline acetyltransferase (CHAT) (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine in the central and peripheral nervous systems. In this study, a human CHAT genomic clone has been isolated and partially sequenced at its 5' end. This fragment contains the first four exons with an ACG initiator codon and potential control regions including TATA, CAAT, GC boxes, and several transcription control sequences. A comparison of the primary structure of CHAT among pig, rat, mouse, and Drosophila is presented.


Molecular cloning of mouse beta 2-glycoprotein I and mapping of the gene to chromosome 11. beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.


Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio). To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 x 10(5) copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitutes 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.


Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF. Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.


The HRAS1 gene cluster: two upstream regions recognizing transcripts and a third encoding a gene with a leucine zipper domain. We have cloned and characterized a 55-kb region of DNA surrounding HRAS1. It contains a cluster of two, and possibly three, genes associated with CpG islands within the 32 kb immediately upstream of HRAS1. We have sequenced cDNAs representing one of these genes, provisionally designated HRC1. The locus, which is located 29 kb upstream of HRAS1, is divergently transcribed. HRC1 cDNA probe recognizes fragments on Southern blots of DNA from other vertebrate species. In human DNA, multiple homologous fragments are detected in addition to the predicted ones containing HRC1. Therefore, this locus may represent a member of an evolutionarily conserved gene family. HRC1 expression is upregulated with HRAS1 in the EJ bladder carcinoma cell line, suggesting the possibility of coordinate regulation. The deduced translational product of the longest open reading frame (1119 nucleotides, 373 amino acids) predicts a protein with regions rich in glutamine and proline and a region similar to the helix-loop-helix motif adjacent to a carboxy-terminal leucine zipper dimerization motif with four heptad repeats. Alternate splicing of terminal exons occurs, resulting in the truncation of one proline-rich domain and preservation of the leucine zipper. Thus, a biologically important region of chromosome 11p consists of a gene cluster. At least one of these genes, in addition to HRAS1, may be involved in regulation of cell growth or differentiation.


Highly repeated DNA sequences in birds: the structure and evolution of an abundant, tandemly repeated 190-bp DNA fragment in parrots. Up to 6.8% of the parrot (Psittaciformes) genome consists of a tandemly repeated, 190-bp sequence (P1) located in the centromere of many if not all chromosomes. Monomer repeats from 10 different psittacine species representing four subfamilies were isolated and cloned. The intraspecific sequence variation ranged from 1.5 to 7%. The interspecific sequence variation ranged from less than 3% between two species of cockatoos to approximately 45% between cockatoos and other parrots. The monomer sequences of all 10 parrot species contained several conserved (> 90%) sequence elements at identical locations within the repeat. A comparison with tandemly repeated DNA sequences in other avian species showed that several of these conserved elements were also present at similar locations within the 184-bp repeat of the Chilean flamingo (Phoenicopterus chilensis), suggesting a great antiquity of the repeat. One of the elements was also found in the tandemly repeated sequences of the crane (Gruidae) and falcon (Falconidae) families. The data were used for the construction of a partial most parsimonious relationship that supports a regional subdivision of the Psittaciformes.


Architecture of the canine IDUA gene and mutation underlying canine mucopolysaccharidosis I. Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficiency of alpha-L-iduronidase. In addition to the well-known human forms (Hurler, Hurler/Scheie, and Scheie syndromes), there exists a canine model of the disease. By using previously described canine cDNA encoding alpha-L-iduronidase as a probe, the canine IDUA gene has been cloned and characterized. It contains 14 exons spread over 13 kb. An unusual GC dinucleotide was found at the donor splice site of intron 11. A transcriptional start site was identified by primer extension 177 bp upstream of the initiator AUG codon. The upstream region was found to be similar to the promoter region of many housekeeping genes: it is GC rich and has seven potential Sp1 binding sites but no TATA box or CAAT motif. The mutation in canine MPS I was localized to the area of intron 1 by RT-PCR, identified by sequence analysis of amplified genomic DNA, and confirmed by restriction analysis; it is a G-->A transition in the donor splice site of intron 1. The mutation causes retention of intron 1 in the RNA and creates a premature termination codon at the exon-intron junction.


Characterization of three VNTR systems at D21S112. D21S112 is a highly polymorphic marker on the long arm of chromosome 21. Our analysis of this locus indicated the presence of three VNTR systems. We estimated the heterozygosity of each system and sequenced one of the repetitive regions. Utilizing PCR, we demonstrated that the sequenced VNTR is responsible for the system with the highest level of heterozygosity. Combining data from the three systems makes D21S112 one of the most informative loci on the chromosome.


Isolation and expression of linked zinc finger gene clusters on human chromosome 11q. Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.


Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal. XY females (n = 17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.


Molecular basis of group A xeroderma pigmentosum: a missense mutation and two deletions located in a zinc finger consensus sequence of the XPAC gene. The molecular basis of group A xeroderma pigmentosum (XP) was investigated, and 3 mutations located in a zinc finger consensus sequence (nucleotide 313-387) of the XP group A complementing (XPAC) gene were identified in 2 Caucasian patients GM2990 and GM2009 who had typical symptoms of group A XP. The first mutation was a C deletion at nucleotide 374. Patient GM2990 was a homozygote for this mutation. The second mutation was a 5-bp deletion (CTTAT) at nucleotides 349-353. The third mutation was a G to T transversion at nucleotide 323 that alters the Cys-108 codon (TGT) to a Phe codon (TTT). Patient GM2009 was a compound heterozygote for the 5-bp deletion and the missense mutation. Both deletions introduce frameshifts with premature translation terminations resulting in instability of the XPAC mRNA and disruption of the putative zinc finger domain of the XPAC protein. The missense mutation also predicts disruption of the zinc finger domain of the XPAC protein. The expression study showed that the missense mutation does indeed causes loss of repair activity of the XPAC protein. We conclude that these 3 mutations are responsible for group A XP.


Human triosephosphate isomerase: substitution of Arg for Gly at position 122 in a thermolabile electromorph variant, TPI-Manchester. Denaturing gradient gel electrophoreses of polymerase chain reaction amplified DNA products and subsequent direct sequencing identified a G-to-A transition causing a replacement of Gly 122 with Arg in an electrophoretic mobility variant of human triosephosphate isomerase, TPI-Manchester. This was the only TPI electromorph variant detected in screening of greater than 3,400 humans in an Ann Arbor, Mich. population. This substitution is at the amino terminus or solvent interaction end of the fifth beta sheet of the alpha/beta barrel structure. The TPI-Manchester variant is a thermolabile enzyme, but the stability of the variant enzyme is not sensitive to other denaturants. This amino acid substitution does not involve residues of the active site and does not detectably alter the kinetic properties of the enzyme. The data provide additional insight into the amino acid residues that are important for the maintenance of the structural characteristics of this very evolutionary constrained protein.


Isolation of DNTR polymorphisms from yeast artificial chromosomes encompassing X chromosomal loci PGK1 and DXS56. Five dinucleotide tandem repeat (DNTR) sequences were isolated from yeast artificial chromosomes containing the PGK1 and DXS56 loci in Xq13. Sequence information of these DNTR loci is given. Four of the five DNTR sequences were polymorphic. Polymorphism information content values were 0.44, 0.49, 0.47, and 0.76 for loci PY5-10, PY2-31, 4548-1, and 4548-7, respectively. Corresponding heterozygosities were 0.55, 0.55, 0.56, and 0.78. These DNTRs are useful for the fine mapping of disease loci in Xq13 and provide sequence tagged sites for this region of the X chromosome.


Alpha-1-antichymotrypsin variant detected by PCR-single strand conformation polymorphism (PCR-SSCP) and direct sequencing. A new mutant alpha-1-antichymotrypsin (variant ACT) was found by polymerase chain reaction single strand conformation polymorphism and direct sequencing. In this variant ACT, two bases (AA) were deleted from codon 391. This resulted in a different amino acid sequence downstream of the deletion point, elongating the peptide chain by 10 amino acids.


A single base deletion from the C1-inhibitor gene causes type I hereditary angio-oedema. RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a C1-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the C1-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of C1-inhibitor gene mutation in type I HAE.


Human C-type natriuretic peptide. Characterization of the gene and peptide. We isolated the human C-type natriuretic peptide gene and identified the peptide in the brain. The human C-type natriuretic peptide gene appeared to be composed of at least two exons and one intron. In the 5'-flanking region, there is an array of cis elements (an inverted CCAAT box, two GC boxes, and a cyclic AMP response element-like sequence) that is not present in upstream sequences of the atrial and brain natriuretic peptide genes. Analysis of the deduced amino acid sequence revealed that human prepro C-type natriuretic peptide comprises 126 amino acids and that the C-terminal 22-residue peptide (G-L-S-K-G-C-F-G-L-K-L-D-R-I-G-S-M-S-G-L-G-C) preceded by Lys-Lys is identical to the porcine counterpart. However, replacement of two amino acids took place in the C-terminal 53-residue sequence, corresponding to another endogenous form of the peptide. Reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for C-type natriuretic peptide demonstrated that it occurs in the human brain. C-type natriuretic peptide-like immunoreactivity was detected in discrete regions of the human brain, and its level was 10-fold higher than the atrial and brain natriuretic peptide levels, raising the possibility that C-type natriuretic peptide is the major natriuretic peptide in the human brain.


DNA polymorphisms in the 5'-flanking region of the HLA-DQA1 gene. The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5' flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5' flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5' end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.


Adherence, coaggregation, and hydrophobicity of Streptococcus gordonii associated with expression of cell surface lipoproteins. Streptococcus gordonii Challis incorporated exogenous [3H]palmitate into 13 polypeptides extractable from intact cells with sodium dodecyl sulfate. A 76-kDa surface-exposed polypeptide, implicated previously as a cell aggregation determinant, was shown to be one of these lipid-modified polypeptides. Differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of lipopolypeptides were detected with mutants of S. gordonii that were altered in adherence, aggregation, coaggregation, or hydrophobicity. Lipid-modified polypeptides, tightly associated with the cell membrane, may be involved in the expression of cell surface properties of S. gordonii important for colonization of the human oral cavity.


The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species. A membrane-associated lipoprotein of Haemophilus influenzae type b has previously been shown to bind heme in vitro and to promote binding of this compound by Escherichia coli recombinants expressing this protein. The H. influenzae type b heme-binding protein A (HbpA) was found to be highly conserved with respect to both antigenicity and apparent molecular weight among heme-requiring Haemophilus species pathogenic for humans. To further the characterization of the structure and function of HbpA, the complete nucleotide sequence of its gene, hbpA, was determined. Analysis of the nucleotide sequence revealed a single large open reading frame of 1,638 bp encoding a protein of 546 amino acid residues, with a molecular weight of 60,695. The sequence of the amino-terminal end of this protein contained a potential site for lipid acylation and for cleavage by signal peptidase II, consistent with earlier biochemical evidence which indicated that HbpA is a lipoprotein. A search of GenBank for proteins with amino acid sequence similarity to HbpA revealed that the periplasmic dipeptide transport protein of E. coli, DppA, has 53% sequence identity to HbpA.


Fungus-specific translation elongation factor 3 gene present in Pneumocystis carinii. Historically, Pneumocystis carinii pneumonia has been the most frequent cause of morbidity and mortality in patients with AIDS. Antiprotozoan drugs are effective in the treatment and prophylaxis of P. carinii pneumonia, which lends credence to the widely held view that P. carinii is a protozoan. However, recent genetic evidence suggests that P. carinii should be classified as a fungus. Translation elongation factor 3 (EF-3) is an essential, soluble translation component which is unique to fungal protein synthesis and is not required for protein synthesis in other eukaryotes. We have identified and isolated a gene for EF-3 from P. carinii, adding more evidence for this organism's assignment as a fungus.


Similarity of Chlamydia pneumoniae strains in the variable domain IV region of the major outer membrane protein gene. DNA was amplified by polymerase chain reaction from the gene encoding the major outer membrane protein (MOMP) of Chlamydia pneumoniae in order to examine the relatedness of strains isolated from diverse geographical regions. Primers for this reaction were chosen to span a 207-bp region comparable to that of the fourth variable segment of the MOMP gene of Chlamydia trachomatis. Among C. trachomatis, sequence heterogeneity is characteristic within variable sequence domain IV (VDIV) and correlates with serovar type. In contrast, sequence analysis of polymerase chain reaction products from 13 C. pneumoniae isolates indicated that all tested strains were identical in this segment of the MOMP gene. The predicted amino acid sequences from the C. pneumoniae VDIV gene products shared only 13.3 to 30% homology with published VDIV regions from serovars of C. trachomatis. Homology of these VDIV amino acid sequences with sequences from strains of C. psittaci ranged from 45.7 to 60%. The sequence conservation of the VDIV region of the MOMP gene indicates that C. pneumoniae strains may be more genetically homogeneous than C. trachomatis or Chlamydia psittaci strains. Future investigations of antigenic diversity among C. pneumoniae strains should be aimed at the evaluation of variation in other regions of the C. pneumoniae genome.


Comparison of the cytolysin II genetic determinants of Actinobacillus pleuropneumoniae serotypes. Cytolysins (Cly) I, II, and III are toxins secreted by Actinobacillus pleuropneumoniae. These toxins are thought to play an important role in the pathogenesis of porcine pleuropneumonia. ClyI and ClyII are RTX toxins and in general these toxins are encoded by operons consisting of four genes, C, A, B, and D. Our group recently cloned the C and A genes of the ClyII operon (clyIICA) of serotype 9. We found that this ClyII operon is truncated and lacked intact B and D genes (clyIIBD). B and D genes of the ClyI operon (clyIBD) were present however in serotype 9. In this study we analyzed the ClyII operons of the reference strains of the 12 A. pleuropneumoniae serotypes and compared them with the ClyII operon of serotype 9. We focused on (i) the presence, (ii) the sequence similarity, and (iii) the genomic environment of the clyIICA genes. The presence of the clyIICA sequences was studied by hybridization analysis of genomic DNA. The sequence similarity was studied by restriction fragment analysis on polymerase chain reaction-amplified DNA. The genomic environment was compared by analysis of the sequences that are located 3' of the clyIICA genes. We demonstrated that the clyIICA genes (i) are present in the reference strains of all serotypes, except serotype 10, (ii) have a high degree of sequence similarity, and (iii) are not contiguous with intact clyIIBD genes. We conclude that the organization and nucleotide sequence of the ClyII operons of A. pleuropneumoniae are very similar. We also studied the presence of clyIBD sequences and found them to be present in the reference strains of all serotypes, except serotypes 3 and 6. Thus, in most serotypes the clyIBD genes may complement the absent clyIIBD genes.


Molecular characterization of the argJ mutation in Neisseria gonorrhoeae strains with requirements for arginine, hypoxanthine, and uracil. Arginine auxotrophs are commonly encountered among clinical isolates of Neisseria gonorrhoeae. Arginine auxotrophs which also require hypoxanthine and uracil (AHU strains) compose a unique set of strains that are highly homogeneous and are believed to be clonally derived. The Arg- phenotype of these strains is due to a lesion in the argJ gene encoding ornithine acetyltransferase. We have cloned the mutant argJ gene from an AHU strain and compared the sequence of this gene to the wild-type argJ gene. The mutant gene contained a 3-bp deletion within a repetitive region of the argJ gene. This mutation was restored to the wild-type sequence in a naturally occurring Arg+ revertant of the AHU strain. This deletion was detected in a wide variety of other AHU strains but not in other ArgJ- strains or in ArgJ+ strains, supporting the theory that AHU strains are clonally derived.


Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC). Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.


Insertion of SMRV-H viral DNA at the c-myc gene locus of a BL cell line and presence in established cell lines. Mutation of the c-myc gene locus has occurred during in vitro culture of the Burkitt lymphoma cell line Namalwa. Gene cloning and sequencing of this c-myc gene locus has revealed the insertion of incomplete copies of the Squirrel Monkey Retrovirus-Human (SMRV-H) proviral genome. Insertions of the SMRV-H genome were seen in all Namalwa cell lines tested, with multiple insertions being seen in some sublines. Viral particles with the morphology of type-D retrovirus, budding from these cells, were seen by transmission EM. New insertions of the genome were produced spontaneously in these cells. The origins of the type-D retrovirus and its implications for workers in the field are discussed.


Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H (beta 2-glycoprotein I). Apolipoprotein H, also known as beta-2-glycoprotein I, was purified from human serum, and antiserum produced to denatured apolipoprotein H detected a cDNA clone from a lambda gt11 library derived from human liver. This cDNA coded for the complete sequence of the mature protein. The cDNA insert, along with a polymerase chain reaction product which extended the 5' end of the message, were subcloned and both strands were sequenced. The apolipoprotein H precursor was found to code for 345 amino acids, 326 of which appear in the mature protein. The deduced amino acid sequence of human apolipoprotein H differs from its rat homologue by the presence of a 48-amino acid stretch which is absent from the rat protein. The remainder of the proteins share a greater than 80% similarity. The amino acid sequence of apolipoprotein H consists largely of repeated units approximately 60 amino acids in length. These repeats are comparable to "sushi structures" found in a large number of diverse proteins, including complement components, receptors and regulators of complement activation, serum proteins, membrane-associated adhesion proteins, and other structural and catalytic proteins. Apolipoprotein H was shown to be transcribed by human hepatoma cell lines Hep 3B and Hep G2, and rat liver by detection of mRNA using northern blot analysis.


Comparative sequence analyses of Epstein-Barr virus nuclear antigen-2 and -3B genes of a fresh Epstein-Barr virus-2 isolate. The nucleotide sequence of Epstein-Barr virus (EBV) nuclear antigen (EBNA)-2 and EBNA-3B regions of an EBV-2 isolate in Japan was analyzed. The deduced amino acid sequences of EBNA-2 and EBNA-3B of the isolate and a prototype EBV-2 strain AG876 were identical, but the number of the repeated sequence in EBNA-3B was variable as that of IR-3 in EBNA-1. The remarkably well-conserved sequence seems to suggest that the structure of the EBNA-2 and EBNA-3B genes of EBV-2 is crucial for their function in virus replication.


Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene. Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C. J. Vlahos and E. E. Dekker, J. Biol. Chem. 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers. The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product. Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E. coli DH5 alpha F'IQ. When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein. All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized. A strain of E. coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene.


Sequence analysis and complementation studies of the argJ gene encoding ornithine acetyltransferase from Neisseria gonorrhoeae. Clinical isolates of Neisseria gonorrhoeae frequently are deficient in arginine biosynthesis. These auxotrophs often have defects in the fifth step of the arginine biosynthetic pathway, the conversion of acetylornithine to ornithine. This reaction is catalyzed by the enzyme ornithine acetyltransferase, which is a product of the argJ gene. We have cloned and sequenced the gonococcal argJ gene and found that it contains an open reading frame of 1,218 nucleotides and encodes a peptide with a deduced Mr of 42,879. This predicted size was supported by minicell analysis. This gene was capable of complementing both Escherichia coli argE and argA mutations and of transforming an ArgJ- strain of N. gonorrhoeae to Arg+. Southern blots were able to detect bands that specifically hybridized to the gonococcal argJ gene in genomic DNA from Pseudomonas aeruginosa but not E. coli, a result that reflects the divergent nature of the arginine biosynthetic pathway in these organisms.


Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids. The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate.


Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.


Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp. strain NS671. DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp. strain NS671. The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids. Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length. DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE. Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not. Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.


Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein. The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.


Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production. The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.


Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone. Cells with a novel mutation that is lethal when the cells are exposed to visible light were isolated from Escherichia coli K-12. The mutation was mapped at 63 min on the linkage map of the E. coli chromosome, and the gene, designated visB, was cloned and sequenced. From its map position and the evidence that the gene product VisB exhibits homology with flavin monooxygenase of Pseudomonas fluorescens, the visB gene was deduced to be identical to the ubiH gene, which is a gene required for the biosynthesis of ubiquinone and is thought to be similar to the gene for flavin monooxygenase. The photosensitive phenotype appears to be due to the accumulation of the substrate for the reaction catalyzed by the visB (ubiH) gene product because other mutations that block earlier steps in the biosynthesis of ubiquinone can reverse the photosensitivity. The accumulated intermediates may produce active species of oxygen in the mutant bacteria upon illumination by visible light, and these active oxygen species may cause the death of the cells by a mechanism similar to that associated with mutations in visA (hemH).