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Structure, expression, and chromosomal location of the human c-fgr gene.

The nucleotide sequence of seven exons of the human c-fgr gene, a cellular homolog of the oncogene of Gardner-Rasheed feline sarcoma virus, was determined. Twenty-six independent genomic clones were obtained from a human gene library with a DNA clone of Y73 avian sarcoma virus oncogene, v-yes, as a probe under relaxed hybridization conditions. Restriction mapping and partial sequence analyses revealed that two of these clones were derived from the c-fgr gene, distinct from the c-yes gene. Interestingly, the splicing points of the c-fgr gene were identical with those of the c-src gene throughout the seven exons, suggesting that the two proto-oncogenes were generated by gene duplication of an ancestral gene containing intervening sequences. On RNA blot hybridization the major transcript was found to be 2.6 kilobase long. Two additional transcripts of 3.5 and 4.7 kilobases were also detected. Furthermore, karyotype analysis of several human-mouse hybrid cells and Southern blot analyses of DNAs of the hybrids with a human c-fgr locus-specific probe showed that this gene is located on chromosome 1.

Animals↗

A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2. c-K-ras-2 genes were cloned from a gene library of Lu-65 and a single point mutation causing a substitution of cysteine for glycine in codon 12 was found by DNA sequencing. It was concluded that the amplification of the c-myc and c-K-ras-2 genes are accompanied by point mutational activation of c-K-ras-2 in the human LGCC Lu-65. This is the first report of multiple gene amplification accompanied by a point mutation of oncogenes in human cancer cells, providing further support for the idea that co-operation of at least two activated cellular oncogenes is required for carcinogenesis.

Amino Acid Sequence↗

Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor.

Human genomic DNA encoding the gamma subunit precursor of the skeletal muscle acetylcholine receptor has been cloned by screening a gene library with a calf cDNA probe and has been subjected to nucleotide sequence analysis. Comparison of the nucleotide sequence of the cloned human genomic DNA with that of the calf cDNA has revealed that the protein-coding sequence of this gene is divided by 11 introns into 12 exons. Evidence is presented to suggest that the human muscle acetylcholine receptor gamma and delta subunit genes are juxtaposed. The primary structure of the gamma subunit precursor of the human muscle acetylcholine receptor has been deduced from the corresponding gene sequence. This polypeptide is composed of 517 amino acids including a hydrophobic prepeptide of 22 amino acids. The gamma subunit of the human muscle acetylcholine receptor, like the alpha subunit of the same receptor as well as the alpha, beta and gamma subunits of its calf counterpart, shares structural features common to all four subunits of the Torpedo electroplax receptor, such as the putative disulphide bridge corresponding to that in the alpha subunit proposed as being in close proximity to the acetylcholine binding site and the four putative, hydrophobic transmembrane segments M1-M4. Thus, the human gamma subunit molecule apparently exhibits the same transmembrane topology as proposed for the fish receptor subunits. The 12 exons seem to correspond to different structural and functional domains of the gamma subunit precursor molecule. Some exons and the protein regions encoded by them are more highly conserved between the mammalian and Torpedo sequences. The pattern of regional homology observed is consistent with the relatively high conservation of the region encompassing the putative disulphide bridge and of the region containing the putative transmembrane segments M1, M2 and M3.

Amino Acid Sequence↗

Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents.

recA-mediated production of pectin lyase (PNL) and the bacteriocin carotovoricin occurs in Erwinia carotovora subsp. carotovora 71 when this organism is subjected to agents that damage or inhibit the synthesis of DNA. The structural gene pnlA was isolated from a strain 71 cosmid gene library following mobilization of the cosmids into a moderate PNL producer, strain 193. The cosmid complemented pnl::Tn5 but not ctv::Tn5 mutations. A constitutive level of PNL activity was detected in RecA+ and RecA- Escherichia coli strains carrying the pnlA+ gene on the high-copy-number plasmid pBluescript SK-. Mappings of Mu dI1734 (Km lac'ZYA) insertions in pnlA and unidirectional deletion analyses allowed localization of the gene to approximately 1.4 kilobases of DNA. A typical pnlA-lacZ transcriptional fusion was inducible in a RecA+ but not a RecA- derivative of strain 71. In contrast, the pnlA-lacZ fusion was not inducible in a RecA+ E. coli strain. DNA sequences homologous to pnlA were detected in E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica strains and in one of four Erwinia rhapontici strains but not in Erwinia chrysanthemi.

Chromosome Mapping↗

Human monoclonal thyroglobulin autoantibodies: epitopes and immunoglobulin genes.

Autoantibodies to thyroglobulin (TgAbs) are common markers of thyroid autoimmunity, but relatively few human monoclonal TgAbs have been described. From a panel of 64 human monoclonal TgAbs (isolated from a thyroid-disease derived combinatorial Ig gene library), we selected seven with unique genetic features for detailed characterization. These TgAbs preferentially recognize native (not denatured) Tg, like serum autoantibodies. Most have high affinities for Tg (dissociation constant 10(-10) to 10(-9) m). Their light (L) chain Ig genes are not unusual, but four of the five heavy (H) chain genes are new. Moreover, one H chain belongs to the small VH2 family, not previously reported for autoantibodies to Tg or thyroid peroxidase. The TgAbs inhibit the binding to Tg of the thyroid donor's serum autoantibodies, indicating epitopic overlap. Competition analysis (surface plasmon resonance) shows that the TgAbs recognize overlapping epitopes in an immunodominant region on the Tg dimer ( approximately 660 kDa). Two major and several minor epitopic regions were defined, each associated with a particular H + L chain combination. In conclusion, our TgAb panel provides novel information regarding the repertoire of H chain genes encoding human TgAbs as well as the relationship between the H chains and the epitopes recognized on this major thyroid autoantigen.

Amino Acid Sequence↗

Isolation of the Thiobacillus ferrooxidans ntrBC genes using a T. ferrooxidans nifH-lacZ fusion.

An agar plating technique was developed in which the activation of expression of a Thiobacillus ferrooxidans nifH-lacZ gene fusion was used to isolate the ntrBC genes from a T. ferrooxidans gene library. An Escherichia coli ntrC mutant containing the nifH-lacZ fusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T. ferrooxidans ntrBC genes. A 4.47 kb region from the T. ferrooxidans chromosome was sequenced. Analysis of the sequence revealed that the ntrB and ntrC genes were closely linked to a third ORF of unknown function. Analysis of the 900 bp region upstream of the T. ferrooxidans ntrBC genes and Southern hybridization experiments confirmed that in T. ferrooxidans ATCC 33020, the glnA and ntrBC genes are unlinked. Expression of the T. ferrooxidans nifH-lacZ fusion in E. coli was activated in the presence of the T. ferrooxidans ntrBC genes and regulated by nitrogen.

Amino Acid Sequence↗

Gene sequence encoding early enzymes of arginine synthesis within a cluster in Bacillus subtilis, as revealed by cloning in Escherichia coli.

From a partial Sau3A gene library of Bacillus subtilis chromosomal DNA in the expression plasmid pRK9, four hybrid plasmids were isolated carrying overlapping segments of the argA-argF-cpa cluster. The complementation patterns within Escherichia coli arginine auxotrophs of these hybrids and deletion derivatives provided the gene order argC-argA-argE-argB-argD-cpa-argF.

Arginine↗

Two gene clusters of Rhizobium meliloti code for early essential nodulation functions and a third influences nodulation efficiency.

A pLAFR1 cosmid clone (pPP346) carrying the nodulation region of the symbiotic plasmid pRme41b was isolated from a gene library of Rhizobium meliloti 41 by direct complementation of a Nod- deletion mutant of R. meliloti. Agrobacterium tumefaciens and Rhizobium species containing pPP346 were able to form ineffective nodules on alfalfa. The 24-kilobase insert in pPP346 carries both the common nodulation genes and genes involved in host specificity of nodulation. It was shown that these two regions are essential and sufficient to determine the early events in nodulation. A new DNA region influencing the kinetics and efficiency of nodulation was also localized on the symbiotic megaplasmid at the right side of the nif genes.

DNA, Bacterial↗

Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression.

Escherichia coli strains defective in the rpiA gene, encoding ribose phosphate isomerase A, are ribose auxotrophs, despite the presence of the wild-type rpiB gene, which encodes ribose phosphate isomerase B. Ribose prototrophs of an rpiA genetic background were isolated by two different approaches. Firstly, spontaneous ribose-independent mutants were isolated. The locus for this lesion, rpiR, was mapped to 93 min on the linkage map, and the gene order zje::Tn10-rpiR-mel-zjd::Tn10-psd-purA was established. Secondly, ribose prototrophs resulted from the cloning of the rpiB gene on a multicopy plasmid. The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage lambda 10H5 (642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B (molecular mass, 16,063 Da) and a negative regulator of rpiB gene expression, RpiR (molecular mass, 32,341 Da), respectively. The 5' ends of rpiB- and rpiR-specified transcripts were located by primer extension analysis. No significant amino acid sequence similarity was found between ribose phosphate isomerases A and B, but ribose phosphate isomerase B exhibited high-level similarity to both LacA and LacB subunits of the galactose 6-phosphate isomerases of several gram-positive bacteria. Analyses of strains containing rpiA, rpiB, or rpiA rpiB mutations revealed that both enzymes were equally efficient in catalyzing the isomerization step in either direction and that the construction of rpiA rpiB double mutants was a necessity to fully prevent this reaction.

Aldose-Ketose Isomerases↗

Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca.

The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.

Amino Acid Sequence↗

Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation.

As a first step in an analysis of the DNA regions involved in the control of the catalase A gene of Saccharomyces cerevisiae by glucose, heme, and oxygen this gene has been cloned. Catalase A-deficient mutants were obtained by UV mutagenesis of a ctt1 mutant strain specifically lacking catalase T. All the catalase A-deficient mutants obtained fall into one complementation group. The single recessive mutation causing specific lack of catalase A was designated cta1. Several overlapping DNA fragments complementing the cta1 mutation were obtained by transforming ctt1 cta1 double mutants with a yeast gene library in vector YEp13. Hybrid selection of RNA with the help of one of the cloned DNAs followed by in vitro translation of this RNA and identification of the protein synthesized with catalase A-specific antibodies showed that the catalase A structural gene has been cloned. A single copy of this gene is present in the yeast genome. Transcription of the catalase A gene cloned into vector YEp13 is repressed by glucose. The DNA isolated hybridizes to a 1.6 kb polyA+-RNA virtually absent from heme-deficient cells, presumably catalase A mRNA.

Alleles↗

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12.

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.

Bacterial Proteins↗

Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455.

Streptomyces noursei ATCC 11455 produces the antifungal polyene antibiotic nystatin containing the deoxysugar moiety mycosamine. Part of the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene (gdhA) known to be involved in deoxysugar biosynthesis was amplified by PCR from genomic DNA of S. noursei ATCC 11455. A gene library for S. noursei was made and screened with the gdhA probe. Several overlapping phage clones covering about 30 kb of the S. noursei genome were physically mapped. A partial DNA sequencing analysis of this region resulted in the identification of several putative genes typical of macrolide antibiotic biosynthetic gene clusters. A gene-transfer system for 5. noursei has been established, and gene deletion or disruption experiments within the putative biosynthetic gene cluster were performed. All of the knock-out mutants retained the ability to produce nystatin, suggesting that the identified gene cluster is not involved in biosynthesis of this antibiotic. Culture extracts from the wild-type strain and three knock-out mutants were analysed by TLC followed by a bioassay against Micrococcus luteus. Two antibacterial compounds were found to be synthesized by the wild-type strain while only one was produced by the mutants. This provided evidence for the involvement of the identified gene cluster in the biosynthesis of a presumably novel antibacterial macrolide antibiotic in S. noursei.

Anti-Bacterial Agents↗

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes.

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.

Bacterial Proteins↗

Cloning, expression, and mapping of the Aeromonas hydrophila aerolysin gene determinant in Escherichia coli K-12.

DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.

Aeromonas↗

Structural organization of rat ribosomal genes restriction endonuclease analysis of genomic and cloned ribosomal DNAs.

Structural organization of the rat ribosomal repeating unit was studied using hybridization of blotted restriction fragments of total rat DNA with alpha-32P-labeled cDNA probe synthesized on the 18S and 28S rRNAs. A detailed restriction endonuclease map was constructed, the 18S and 28S rRNA genes mapped and the sizes of the rat ribosomal repeating units determined. Considerable site heterogeneity of rat rDNA was revealed in both nontranscribed and external transcribed spacers. Recombinant phages containing the whole set of transcribed regions and a considerable part of a nontranscribed spacer of the rDNA were selected from the rat gene library. The restriction maps of the cloned rDNA fragments are in good agreement with the map constructed by Southern's technique, add to this map and support the existence of site heterogeneity in ribosomal repeating units. Proximal to the 3'-end of the 28S rRNA gene an internally repetitive region was found, each repeating unit being equal to approx. 150 bp. The site for transcription initiation was mapped 4.0-4.5 kb upstream from the 5'-end of the 18S rRNA gene. Frequently reiterated interspersed sequences were found in the nontranscribed spacer at approx. 2-3 kb distance from both ends of the transcribed region.

Animals↗

A large-scale study of Yap1p-dependent genes in normal aerobic and H2O2-stress conditions: the role of Yap1p in cell proliferation control in yeast.

Yeast genes regulated by the transcriptional activator Yap1p were screened by two independent methods: (i) use of a LacZ-fused gene library and (ii) high-density membrane hybridization. Changes in transcriptome profile were examined in the presence and in the absence of Yap1p, as well as under normal and H2O2-mediated stress conditions. Both approaches gave coherent results, leading to the identification of many genes that appear to be directly or indirectly regulated by Yap1p. Promoter sequence analysis of target genes revealed that this regulatory effect is not always dependent upon the presence of a Yap1p binding site. The results show that the regulatory role of Yap1p is not restricted to the activation of stress response but that this factor can act as a positive or a negative regulator, both under normal and oxidative stress conditions. Among the targets, a few genes participating in growth control cascades were detected. In particular, the RPI1 gene, a repressor of the ras-cAMP pathway, was found to be downregulated by Yap1p during the early phase of growth, but upregulated in the stationary phase or after oxidative stress.

Aerobiosis↗

Molecular cloning of the gene encoding CDPdiacylglycerol-inositol 3-phosphatidyl transferase in Saccharomyces cerevisiae.

The structure gene encoding CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated by complementation in a Saccharomyces cerevisiae mutant after transformation with a comprehensive gene library containing Sau3AI partial restriction fragments of wild-type yeast DNA inserted into the YEp13 shuttle vector. Introduction of the cloned plasmid into the mutant restored both growth of cells and CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity. A subcloning study indicated that the complementing gene was contained within a 1700-base-pair fragment of DNA. Expression of the cloned sequence was independent of its orientation in the vector. CDPdiacylglycerol-inositol 3-phosphatidyltransferase activity in the transformant was eight fold higher than that in the wild-type strain. Although the mutant enzyme had a greatly increased Km for myo-inositol, the enzyme in the transformant had an apparent Km for myo-inositol equal to that of the wild-type enzyme, indicating the presence of the wild-type structure gene on the recombinant plasmid. The elevated level of enzyme activity in the transformant did not lead to an increase in the content of phosphatidylinositol. In contrast, supplementation of the culture medium with increasing concentrations of myo-inositol resulted in an increase in the phosphatidylinositol content. Thus, the availability of myo-inositol is a critical regulatory factor in yeast phosphatidylinositol synthesis.

Base Composition↗