Isolation of a hagfish gene that encodes a complement component.

It has been widely accepted that cyclostomes are the most primitive vertebrates extant with the ability to produce antibodies. We isolated cDNA clones that encode a putative 'antibody' from one of the cyclostomes, Eptatretus burgeri. The amino acid sequence predicted from the nucleotide sequences of the cDNA clones indicated that this gene does actually encode the proteins isolated as hagfish 'antibodies' by various investigators. However, these proteins are not similar to mammalian immunoglobulins but have some characteristics common to complements C3, C4 and C5 in higher vertebrates. We discuss the relationships of the isolated gene for hagfish complement with the mammalian genes for complements C3, C4 and C5. We also discuss the possibility of the presence of antibodies in cyclostomes.     

Isolation of a maltase structural gene from Saccharomyces carlsbergensis.

The maltase structural gene MAL6 of the yeast Saccharomyces carlsbergensis has been cloned by transformation of a maltose nonfermenting recipient strain with autonomously replicating chimeric recombinant plasmids. One recombinant plasmid, pMAL26, was shown by positive hybridization translation, as well as by Southern and Northern blot experiments, to carry the MAL6 structural gene.     

Isolation and structure of a yeast initiator tRNAmet gene.

Sixteen bacterial clones containing yeast initiator tRNAmet genes have been isolated. The size of the BamHI fragments encoding these genes ranges from 4,000 to 23,000 base pairs. The nucleotide sequence of one member of this group has been determined. It has no intervening sequences.     

Isolation and oncogenic potential of a novel human src-like gene.

We have isolated cDNA molecules representing the complete coding sequence of a new human gene which is a member of the src family of oncogenes. Nucleotide sequence analysis revealed that this gene, termed slk, encoded a 537-residue protein which was 86% identical to the chicken proto-oncogene product, p60c-src, over a stretch of 191 amino acids at its carboxy terminus. In contrast, only 6% amino acid homology was observed within the amino-terminal 82 amino acid residues of these two proteins. It was possible to activate slk as a transforming gene by substituting approximately two-thirds of the slk coding sequence for an analogous region of the v-fgr onc gene present in Gardner-Rasheed feline sarcoma virus. The resulting hybrid protein molecule expressed in transformed cells demonstrated protein kinase activity with specificity for tyrosine residues.     

Isolation and nucleotide sequence of a mouse histidine tRNA gene.

We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T residues. 523 residues after the 3' end of the his tRNA coding region, the mouse DNA contains a sequence 72% homologous to part of the consensus sequence of the B1 (alu) family.     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

Isolation and structure of a rhodopsin gene from D. melanogaster

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.     

Isolation of a yeast gene involved in species-specific pre-tRNA processing.

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.     

Isolation and sequence analysis of a novel human tyrosine kinase gene.

Using v-abl probes, we have identified and cloned a novel fes/fps-homologous human cDNA, which we have designated FER (pronounced "fair"). This apparently full-length cDNA of 3.0 kilobases has an open reading frame of 2,466 base pairs and the capacity to encode a protein of 94,000 molecular weight. The cDNA contains regions homologous to the highly conserved tyrosine protein kinase domain of other oncogenes and growth factor receptors but lacks a clear transmembrane region, indicating that it encodes a tyrosine kinase of the nonreceptor type. The deduced amino acid sequence of FER resembles that of c-fes/fps. Our data indicate that the protein product of FER, p94FER, corresponds to a previously reported cellular phosphoprotein, NCP94, detected with a v-fps-specific antipeptide antiserum.     

Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.     

Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.