Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

Cloning and characterization of the aldA gene of Aspergillus nidulans

We have cloned and sequenced the aldA (encoding aldehyde dehydrogenase) gene of Aspergillus nidulans. The gene contains two introns which are similar in size and structure to other fungal introns. The amino acid sequence of aldehyde dehydrogenase (497 residues) shows a significant level of homology with analogous sequences in other organisms. Comparison of the primary structure of the active sites of the mammalian cytosolic and mitochondrial enzymes shows that the Aspergillus enzyme closely resembles the mammalian mitochondrial enzyme. Analysis of the 5' non-coding region of the aldA gene shows a TATA-like sequence located 90 bp upstream from the initiation codon. Two messenger-RNA start points are located 36 and 42 bp upstream from the start codon.     

Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage.

We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries. The human gene contains 15 introns and is 7.1 kb long. The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions. The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3.     

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene,a homologue of the ras protooncogene

We have cloned the genomic copy of the Pprasl gene, a homologue of the ras proto-oncogene, from the true slime mold Physarum polycephalum. Pprasl contains five small introns, four of which have a high content of pyrimidines. The (dC)-homopolymers present in introns 4 and 5 may be responsible for the observed recA-independent deletion in Pprasl upon amplification of the Pprasl-bearing plasmid by chloramphenicol. Although Pprasl exhibits high amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Dictyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently     

The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts.

Using cloned DNA complementary to mouse dihydrofolate reductase (DHFR) mRNA, the organization of the hamster DHFR gene has been determined in two baby hamster kidney (BHK) cell lines, A5 and B1. A5 cells are highly methotrexate-resistant, containing 200-fold more copies of the DHFR gene than do the parental B1 cells. The DHFR gene has the same organization in A5 and B1 cells, suggesting that it has not been altered by the amplification process. The BHK DHFR gene spans a maximum of 10.7 kb and contains at least three introns. Thus the BHK DHFR gene is much smaller than the mouse DHFR gene, which has a minimum size of 42 kb and at least five introns. This striking size difference is probably due to much smaller introns in the BHK DHFR gene.     

Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene

The existence of a neurofilament-deficient mutant of Japanese quail was recently documented (Yamasaki, H., C. Itakura, and M. Mizutani. 1991. Acta Neuropathol. 82:427-434), but the genetic events leading to the neurofilament deficiency have yet to be determined. Our molecular biological analyses revealed that the expression of neurofilament-L (NF- L) gene was specifically repressed in neurons of this mutant. To search for mutation(s) responsible for the shutdown of this gene expression, we cloned and sequenced the NF-L genes in the wild-type and mutant quails. It is eventually found that the NF-L gene in the mutant includes a nonsense mutation at the deduced amino acid residue 114, indicating that the mutant is incapable of producing even a trace amount of polymerization-competent NF-L protein at any situation. The identification of this nonsense mutation provides us with a solid basis on which molecular mechanisms underlying the alteration in the neuronal cytoskeletal architecture in the mutant should be interpreted.     

The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence

We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase expressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six introns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals.     

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes.

Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb (COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5' end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human x Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.     

A scytalone dehydratase gene from<Emphasis Type="Italic"> Ophiostoma floccosum</Emphasis> restores the melanization and pathogenicity phenotypes of a melanin-deficient<Emphasis Type="Italic"> Colletotrichum lagenarium</Emphasis> mutant

Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning. Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases. The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported. Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity. The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis. These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C. lagenarium.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.