Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.     

The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms.

Nucleotide (nt) sequences encoding the elongation factor Tu (EF-Tu), tRNA(Thr) and tRNA(Trp) from Chlamydia trachomatis have been determined. The environment of the EF-Tu-encoding gene (tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus. The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies. Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies. Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root. The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences. Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein.     

The sequence of a porcine cDNA encoding α-lactalbumin

A cDNA clone encoding porcine α-lactalbumin (αLA) was isolated and sequenced. The longest clone was 688 nucleotides (nt) long and encoded a preprotein of 141 amino acids (aa) including a leader peptide of 19 aa. The porcine cDNA exhibited a nt similarity of between 72.2%–83.5% to other αLA cDNAs and an aa similarity of between 50.8%–85.2% with other αLA aa sequences. The derived aa sequence varied at three positions from a previously reported sequence for porcine αLA obtained by direct aa sequencing.     

Sequence analysis of mouse cDNAs encoding ribosomal proteins L12 and L18

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a M_r, of 17 790, while L18 has 188 aa and a M_r of 21 570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.     

Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli.

The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level. The order of conservation at the nt and aa level is rpsL greater than tufA greater than rpsG greater than f usA. A striking difference is that the rpsG-encoded ribosomal protein, S7, in E. coli K-12 is 23 aa longer than in S. typhimurium. The very low (0.18) codon adaptation index of this part of the E. coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension. A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S. typhimurium is noted. In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events. An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

Sequence of human eosinophil-derived neurotoxin cDNA: identity of deduced amino acid sequence with human nonsecretory ribonucleases

Several clones of human eosinophil-derived neurotoxin (EDN) cDNA have been isolated from a lambda gt10 cDNA library prepared from mRNA derived from noninduced HL-60 cells. The amino acid (aa) sequence deduced from the coding sequence of the EDN cDNA is identical to the aa sequence of urinary nonsecretory RNase. Comparison of the aa and/or nucleotide (nt) sequences of EDN and other proteins possessing ribonucleolytic activity, namely bovine seminal RNase, human and rat pancreatic RNases, eosinophil cationic protein (ECP), and human angiogenin, shows extensive identity at half-cystine residues and at aa of active sites. Differences in aa sequences at the active sites are often the result of single nt changes in the codons. The data presented here support the concept of a RNase gene superfamily containing secretory and nonsecretory RNases, angiogenin, EDN and ECP.     

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1α, EF-1β, EF-1γ, EF-2 and EF-3. With the exception of the EF-1γ gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1α, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3′ untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1α genes are located at different loci. Northern analysis indicated that one of three EF-1α genes was inducible with UV-irradiation, while the level of expression for another of three EF-1α genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1β cDNA clone covered almost all the coding sequence (CDS) of EF-1β except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3′ UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.     

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.     

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject     

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

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.     

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus,Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.