Cytoplasmic Leucyl-Trna Synthetase of Neurospora Crassa Is Not Specified by the Leu-5 Locus

We generated a lambda gt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, lambda NCLRSC1 and lambda NCLRSC2, were obtained which have inserts of approximately 2 kbp and approximately 1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that lambda NCLRSC1 and lambda NCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by lambda NCLRSC1 or lambda NCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an approximately 115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is approximately 3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.     

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.     

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.     

Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.     

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Molecular cloning and characterization of cDNA for human myeloperoxidase

Partial amino acid sequence of human myeloperoxidase was determined, and a 41-base oligonucleotide containing deoxyinosines at four positions was chemically synthesized. By using the oligonucleotide as a probe, cDNA clones for human myeloperoxidase were isolated from a cDNA library constructed with mRNA from human promyelocytic leukemia HL-60 cells. One of the clones containing a 2.6-kilobase insert was subjected to nucleotide sequence analysis. The sequence was found to contain an open reading frame, 2,235 nucleotides coding for a protein of 745 amino acids with a calculated Mr of 83,868. The heavy chain of myeloperoxidase, consisting of 467 amino acids, was located on the COOH terminus half of the protein. The RNA specified by the cDNA was prepared using SP6 RNA polymerase and translated in rabbit reticulocyte lysates, and the product was identified as human myeloperoxidase by immunoprecipitation with rabbit anti-human myeloperoxidase antibody. By Northern hybridization analysis of RNA from leukemic cells, it was shown that myeloperoxidase mRNA is abundantly expressed in human promyelocytic HL-60 and mouse myeloid leukemia NFS-60 cells. Furthermore, the results of Southern hybridization analysis of human genomic DNA suggest that there are one or two genes for myeloperoxidase in the human haploid genome.     

Identification of recombinant phages containing sequences from different rat myosin heavy chain genes.

The construction and identification of a recombinant plasmid containing a cDNA insert which hybridizes specifically to myosin heavy chain mRNA is described. The plasmid was used as a probe to screen a rat genomic library for recombinant phages containing myosin heavy chain sequences. Six clones with approximately 15 k bp inserts each were isolated. Digestion with several restriction enzymes and hybridization of the fractionated DNA with the plasmid probe showed that the clones contained 3 different DNA inserts. Electron microscopy of a heteroduplex made by hybridization of DNA from two clones confirmed that the inserts originated in different genes. Hybridization of size-fractionated ECOR1 digested rat spleen DNA with the cloned probe suggested the existence of at least 5 myosin heavy chain genes.     

Primary structure of human pepsinogen gene

A recombinant clone, which covers the pepsinogen gene in a single insert, has been isolated by screening a library of human genomic DNA, using a swine pepsinogen cDNA as a probe. Sequence analysis of coding DNA segments of the clone revealed that the pepsinogen gene occupies approximately 9.4-kilobase pairs of the genomic DNA and is separated into nine exons by eight introns of various lengths. The predicted amino acid sequence of human pepsinogen consists of 373 residues and is 82% homologous with that of swine pepsinogen. In addition, the predicted sequence contained a single sequence of 15 amino acid residues at the NH2 terminus, showing that the protein is synthesized as prepepsinogen. The structure of the gene, in which two homologous sequences including the two active site aspartyl residues of pepsin are present in different coding segments, is in support of the view that the pepsinogen gene evolved by duplication of a shorter ancestral gene.     

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5'-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.     

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

We have isolated genomic clones for human fibronectin (FN), by screening a human gene library with previously isolated FN cDNA clones. We have recently reported two different FN mRNAs, one of them containing an additional 270 nucleotide insert coding for a structural domain ED. Restriction mapping and DNA sequencing of the genomic clones show that the ED type III unit corresponds to exactly one exon in the gene, whilst the two flanking type III units are split in two exons at variable positions. When an alpha-globin/FN gene hybrid construct, containing the ED exon, flanking introns and neighbouring FN exons, is transfected into HeLa cells, two hybrid mRNAs differing by the ED exon are synthesized. These experiments confirmed that the two FN mRNAs observed in vivo arise from the same gene by alternative splicing.