Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.     

Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108.

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.     

Primary structure of the alanine carrier protein of thermophilic bacterium PS3.

Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.     

Primary structure of non-histone chromosomal protein HMG2 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone for the entire sequence of pig thymus non-histone protein HMG2 are described. cDNA the size of 1153 nucleotides contains an open reading frame of 627 nucleotides. The 5'-untranslated region of 146 nucleotides is extremely rich in GC residues whereas the 3'-untranslated region of 380 nucleotides is rich in AT residues. The open reading frame encodes 209 amino acids, which contain a unique continuous run of 23 acidic amino acids at the C-terminal. The deduced amino acid sequence is 79% homologous to that of HMG1 protein from the same source which we reported [Tsuda, K., Kikuchi, M., Mori, K., Waga, S., & Yoshida, M. (1988) Biochemistry 27, 6159-6163]. In addition, the hydropathy index profiles of both proteins are very similar, supporting that they have similar structural features. Northern analysis of poly(A+) RNA reveals that a single-sized mRNA codes for HMG2 protein. Southern analysis suggests that the HMG2 coding gene is homogeneous within the pig thymus genome.     

Cloning and nucleotide sequence of the chimpanzee c-myc gene

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.     

Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure

Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain alpha-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases.     

cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.     

Purification,cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-11: comparison to the human sequence

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-11. Sequence analysis indicated that the monkey apo C-11 cDNA was 200 by longer than the human and the difference in size was all in the 5° untranslated region of the mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-11 cDNA sequence encoding a preprotein of 101 amino acids — identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-11 amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-11 and this is confirmed by isoelectric focusing and immunoblotting.     

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.     

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

Cloning and sequence analysis of the Candida utilis HIS3 gene

A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA library by complementation of the E. coli hisB mutant. Its nucleotide sequence was determined and it predicts a single open reading frame of 675 bp (224 aa). The deduced amino acid sequence is highly homologous to other yeast and fungi HIS3 genes.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.     

Cloning and sequencing of the gene coding for the large subunit of methylamine dehydrogenase from Thiobacillus versutus.

The gene that codes for the alpha-subunit of methylamine dehydrogenase from Thiobacillus versutus, madA, was cloned and sequenced. It codes for a protein of 395 amino acids preceded by a leader sequence of 31 amino acids. The derived amino acid sequence was confirmed by partial amino acid sequencing. The start of the mature protein could not be determined by direct sequencing, since the N terminus appeared to be blocked. Instead, it was determined by electrospray mass spectrometry. Confirmation of the results was obtained by sequencing the N terminus after pyroglutamate aminopeptidase digestion. The sequence is homologous to the Paracoccus denitrificans nucleotide sequence. A second open reading frame, called open reading frame 3, is located immediately downstream of madA.     

The complete primary structure of the human snRNP E protein.

The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.     

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.