Genetic regulation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

A large number of quinic acid non-utilizing qut mutants of Aspergillus nidulans deficient in the induction of all three quinic acid specific enzymes have been analysed. One class the qutD mutants, are all recessive and are non-inducible at pH 6.5 due to inferred deficiency in a quinate ion permease. Two regulatory genes have been identified. The QUTA gene encodes an activator protein since most qutA mutants are recessive and non-inducible although a few fully dominant mutants have been found. The QUTR gene encodes a repressor protein since recessive mutations are constitutive for all three enzyme activities. Rare dominant non-inducible mutants which revert readily to yield a high proportion of constitutive strains are inferred to be qutR mutants defective in binding the inducer. The gene cluster has been mapped in the right arm of chromosome VIII in the order: centromere - greater than 50 map units - ornB - 12 map units - qutC (dehydratase)-0.8 map units-qutD (permease), qutB (dehydrogenase), qutE (dehydroquinase), qutA (activator)-4.4 map units - qutR (repressor)-20 map units - galG. This organization differs from that of the qa gene cluster in Neurospora crassa, particularly in the displacement of qutC and qutR.     

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

BSMAP, a novel protein expressed specifically in the brain whose gene is localized on chromosome 19p12.

Using the sequence of cosmids derived from chromosome 19p12, we have identified a gene encoding a novel protein, BSMAP (brain-specific membrane-anchored protein) and cloned cDNA encoding the full-length open reading frame. Northern blot analysis revealed that BSMAP mRNA is preferentially expressed at a high level in the brain. BSMAP has a putative transmembrane domain and is predicted to be a type-I membrane glycoprotein. Genomic sequence analysis revealed that the gene encoding BSMAP consists of eight exons spanning approximately 8 kb and lies 6 kb away from the gene encoding CLF-1 in a reverse orientation. Although no candidate genetic disorders were found to map either to this precise region of chromosome 19 or to the syntenic region of the mouse genome, the highly specific expression of BSMAP mRNA suggests a role for the protein in CNS function.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.     

Isolation and nucleotide sequence of the ribosomal protein S16-encoding gene from Aspergillus nidulans.

A genomic clone has been isolated from Aspergillus nidulans which is homologous to the ribosomal (r) protein S16-encoding gene of Saccharomyces cerevisiae (S16A) and the r-protein S19-encoding gene of rat (S19). The amino acid (aa) sequences, deduced from nucleotide (nt) sequence analysis, show that in both cases more than 63% of the aa are conserved. The proposed A. nidulans r-protein S16 gene (rps16) differs from that of S. cerevisiae in that it occurs as a single copy in the haploid genome (rather than two copies as in yeast) and contains two putative introns (rather than one). The mRNA leader is long compared to many Aspergillus genes, commencing 293 nt upstream from the coding region, and contains an open reading frame of 13 codons.     

Molecular characterization of the mouse ribosomal protein S24 multigene family: a uniquely expressed intron-containing gene with cell-specific expression of three alternatively spliced mRNAs.

A family of 16 genes encoding the mouse ribosomal protein S24 was identified, and four members from this family were cloned. A single expressed intron-containing S24 gene (termed mrpS24) and one pseudogene (mrpS24p) were completely sequenced and characterized. The mrpS24 gene has seven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt). The cap site of S24 was mapped to a G residue four nt upstream of a polypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element. The 5' region (-325 to +33) of the mrpS24 gene has a functional promoter that was able to express the fused chloramphenicol acetyltransferase (CAT) reporter gene. Two different forms of mouse S24 cDNA clones were previously isolated. Sequence analysis showed that one of these cDNA clones (termed S24a) lacks the entire exon V sequence (18 nt), and the deduced amino acid sequence is missing a C-terminal lysine residue encoded by the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bp direct repeat, and its sequence is almost identical to the S24 cDNA sequence, but it lacks two mini-exons, V and VI (20 nt), as in the cases of the human and rat S24 cDNAs. RT-PCR experiments demonstrated the existence of a third form (S24c) that similarly lacks both of the mini-exons, and suggested that different species of S24 mRNA might arise from alternative splicing of the mini-exons V and VI. Northern blot analysis showed that S24 expression is down- and up-regulated during adipocyte differentiation and in cellular transformation, respectively. RNase protection assays and RT-PCR experiments suggested that these cell-specific changes of S24 mRNA levels are mainly due to fluctuations in S24c mRNA level. Our results provide the first indication that a ribosomal protein gene is regulated by alternative usage of two mini-exons in a cell-specific manner.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides

The trichodiene synthase gene (Tox5) has been isolated from the fungus Fusarium sporotrichioides, and its nucleotide (nt) sequence determined. A lambda gt11 library of F. sporotrichioides DNA was screened with antiserum against trichodiene synthase (TS). DNA fragments were isolated which encode a portion of the Tox5 gene. In subsequent screening of the library we employed one of these DNAs as a probe and identified several recombinant phage containing the entire Tox5 gene. The gene consists of a 1182-nt open reading frame (ORF) which contains a 60-nt intron and specifies a Mr 43,999 protein. The deduced amino acid sequence of the ORF was identical to sequences determined for several CNBr peptides from purified TS. Southern and Northern analyses indicated that the Tox5 gene is present in a single copy and is transcribed into an mRNA of about 1450 nt. Upstream from the start codon, 'TATA'-like sequences and a short repeated sequence resembling the 'CCAAT' box were observed. The primary structure described for TS is the first such report for a member of the terpene cyclase group of enzymes.     

Nucleotide sequence of Lymantria dispar nuclear polyhedrosis virus polyhedrin gene

The polyhedrin gene of the nuclear polyhedrosis virus of the gypsy moth (Lymantria dispar) (LdMNPV) was cloned and sequenced. A polyhedrin open reading frame of 735 nucleotides (nt) was identified which can code for a protein of 245 amino acids that demonstrates a high degree of similarity to other polyhedrins. The protein predicted from the nucleotide sequence shows differences in several regions to that previously sequenced from the LdMNPV polyhedrin protein. The consensus sequence AATAAGTATTTT found at the mRNA start site of baculovirus hyperexpressed genes was located 55 nt upstream from the translational start site.     

Identification of a glialblastoma cell differentiation factor-related gene mRNA in human microvascular endothelial cells

Vascular endothelial cells (VEC) transduce mitogenic and chemoattractant signals in response to erythropoietin (Epo). An analysis of changes in gene expression in VEC would be helpful to understanding the molecular nature of mitogenic signals. An effective method for analysis of gene expression is through differential display. Using this approach, we obtained from Epo-treated human microvascular endothelial cells (HMVEC) a cDNA fragment with characteristics of the 3'end of mRNA. Using the cDNA fragment, we then isolated a full-length clone from a HMVEC cDNA library. The cDNA of interest encodes a protein consisting of 404 amino acids with a carboxy-terminal end sequence identical to glialblastoma cell differentiation factor-related protein (GBDR1). Northern blot analysis showed that GBDR1 mRNA was ubiquitously expressed in human tissues. In Southern blot analysis, GBDR1 cDNA identified a single gene on chromosome 9. Since analysis of the amino acid sequence revealed several putative phosphorylation sites for different protein kinases, the GBDR1 protein was expressed and purified from bacterial extracts and, as predicted, casein kinase II phosphorylated GBDR1 in vitro. Immunofluorescence and biochemical data revealed that the GBDR1 protein is not entirely localized in the cytosolic fraction, suggesting that it may interact with another protein(s). These findings demonstrate that GBDR1 is an intracellular signaling molecule that may play a role in the regulation of endothelial cell growth.