Molecular cloning and characterization of a full-length cDNA clone for human plasminogen

A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24-nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra Ile at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.     

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.     

First Record of HAdV-D20 Among Keratoconjunctivitis Patients in Iraq

Background:Human Adenovirus species D (HAdV-D) was common human viral pathogen especially in eye infection, consists of several types of which HAdV-D8, -D19 and –D37 were common in eye infection. This study includes detection of HAdV-D types implicated in conjunctivitis based on L2 (Penton protein) gene similarity. Methods:Conjunctival swabs were collected from Keratoconjunctivitis patients as eye infection related to adenovirus. Viral nucleic acids were extracted and specific primer pairs for HAdV-D L2 gene (encoding for penton base protein) was used to amplify the target gene and only positive samples were sent to sequencing.Results:The results revealed that only 6 samples give positive results for L2 gene amplification and then sent for sequencing for L2 (penton protein) gene-based typing. The results show that 4 local isolates (S1, S2, S3, S6) were similar to HAdV-D8 and 2 local isolates (S4, S5) were similar to HAdV-D20. Also the results display that the HAdV-37, prominent HAdV-D type of human eye infection, may be variant of HAdV-D20 due to that six variation were seen in S4and S5 local isolates nucleotide sequence in relation to HAdV-D37: T>C at position 14364, A>C at position 14411, T>C at position 14427, C>A at position 14448, G>A at position 14540 and T>C at position 14617, leading to only 2 amino acid change in resulted penton protein: T (Threonine) instead of K (Lysine) at position 204 and N (Asparagine) instead of D (Aspartic acid) at position 247.Conclusion:The current study concludes the possibility of implication of HAdV-D20 in eye infections especially conjunctivitis.Key Words: HAdV-D8, HAdV-D20, HAdV-D37, Conjunctivitis, Iraq     

SNPs occur in regions with less genomic sequence conservation

Rates of SNPs (single nucleotide polymorphisms) and cross-species genomic sequence conservation reflect intra- and inter-species variation, respectively. Here, I report SNP rates and genomic sequence conservation adjacent to mRNA processing regions and show that, as expected, more SNPs occur in less conserved regions and that functional regions have fewer SNPs. Results are confirmed using both mouse and human data. Regions include protein start codons, 3' splice sites, 5' splice sites, protein stop codons, predicted miRNA binding sites, and polyadenylation sites. Throughout, SNP rates are lower and conservation is higher at regulatory sites. Within coding regions, SNP rates are highest and conservation is lowest at codon position three and the fewest SNPs are found at codon position two, reflecting codon degeneracy for amino acid encoding. Exon splice sites show high conservation and very low SNP rates, reflecting both splicing signals and protein coding. Relaxed constraint on the codon third position is dramatically seen when separating exonic SNP rates based on intron phase. At polyadenylation sites, a peak of conservation and low SNP rate occurs from 30 to 17 nt preceding the site. This region is highly enriched for the sequence AAUAAA, reflecting the location of the conserved polyA signal. miRNA 3' UTR target sites are predicted incorporating interspecies genomic sequence conservation; SNP rates are low in these sites, again showing fewer SNPs in conserved regions. Together, these results confirm that SNPs, reflecting recent genetic variation, occur more frequently in regions with less evolutionarily conservation.     

Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system.

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3' non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.     

Multisequence comparisons in protein coding genes. Search for functional constraints

A very powerful method for detecting functional constraints operative in biological macromolecules is presented. This method entails performing a base permanence analysis of protein coding genes at each codon position simultaneously in different species. It calculates the degree of permanence of subregions of the gene by dividing it into segments, c codons long, counting how many sites remain unchanged in each segment among all species compared. By comparing the base permanence among several sequences with the expectations based on a stochastic evolutionary process, gene regions showing different degrees of conservation can be selected. This means that wherever the permanence deviates significantly from the expected value generated by the simulation, the corresponding regions are considered "constrained" or "hypervariable". The constrained regions are of two types: alpha and beta. The alpha regions result from constraints at the amino acid level, whereas the beta regions are those probably involved in "control" processing. The method has been applied to mitochondrial genes coding for subunit 6 of the ATPase and subunit 1 of the cytochrome oxidase in four mammalian species: human, rat, mouse, and cow. In the two mitochondrial genes a few regions that are highly conserved in all codon positions have been identified. Among these regions a sequence, common to both genes, that is complementary to a strongly conserved region of 12S rRNA has been found. This method can also be of great help in studying molecular evolution mechanisms.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

An alpha-1 antitrypsin genetic variant from India

The highly polymorphic human alpha-1 antitrypsin (AAT) gene codes for the most abundant circulating plasma serine protease inhibitor. Previously, genetic variants of the AAT gene were reported from different regions of the world. In the present study, the AAT gene was characterized in an Indian sample. The AAT gene was isolated and cloned from a liver biopsy sample through RT-PCR and the full-length gene was sequenced. Nucleotide sequence comparison with the human genome and the AAT sequences available in the GenBank (NCBI) demonstrated four unique variations--(i) an A to G variation at position 286 (Thr96Ala), (ii) an A to G variation at position 839 (Asp280Gly), (iii) a T to C variation at position 1182 that did not result in any change in the protein sequence (TTT to TTC both code for Phe) and (iv) an A to C variation at position 1200 (Glu400Asp) that resulted in replacement by an amino acid of similar nature. Other variations found were T to C at position 710 (Val273Ala) and T to C position 863 (Val288Glu), which were also reported earlier. In conclusion, this study reports the entire 1257 bp nucleotide sequence of protein coding region of the human AAT gene from an Indian sample. This preliminary finding is significant, as it reports for the first time the AAT gene sequence in the Indian sample.     

Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro

Yeast DNA coding for nucleosome assembly protein I (NAP-I), which facilitates nucleosome assembly in vitro at physiological ionic conditions, was cloned and its gene product was characterized. A monoclonal antibody against NAP-I (58 kDa) from human HeLa cells was used to screen a genomic library of Saccharomyces cerevisiae constructed into lambda gt11. A 60-kDa protein was detected by immunoblotting in the extracts of Escherichia coli lysogenized with a positive clone. The 60-kDa protein purified from the extracts had an activity equivalent to that of NAP-I from mouse and human cells. The amino acid sequence deduced from the gene coding for the yeast NAP-I defines a polypeptide of molecular mass 47,848 Da with three negatively charged regions. While the two regions contain 8 and 10 acidic amino acids out of 13 amino acid residues, the longest stretch has 15 glutamic and 13 aspartic acids out of 38 residues. These regions are probably involved in the interaction with histones. Proteins recognized by the anti-NAP-I antibody were also present in Xenopus oocytes and Drosophila cultured cells. Possible roles of NAP-I are discussed in relation to other nucleosome assembly proteins.     

Multisequence comparisons in protein coding genes

A very powerful method for detecting functional constraints operative in biological macromolecules is presented. This method entails performing a base permanence analysis of protein coding genes at each codon position simultaneously in different species. It calculates the degree of permanence of subregions of the gene by dividing it into segments,c codons long, counting how many sites remain unchanged in each segment among all species compared. By comparing the base permanence among several sequences with the expectations based on a stochastic evolutionary process, gene regions showing different degrees of conservation can be selected. This means that wherever the permanence deviates significantly from the expected value generated by the simulation, the corresponding regions are considered “constrained” or “hypervariable”. The constrained regions are of two types: α and β. The α regions result from constraints at the amino acid level, whereas the β regions are those probably involved in “control” processing. The method has been applied to mitochondrial genes coding for subunit 6 of the ATPase and subunit 1 of the cytochrome oxidase in four mammalian species: human, rat, mouse, and cow. In the two mitochondrial genes a few regions that are highly conserved in all codon positions have been identified. Among these regions a sequence, common to both genes, that is complementary to a strongly conserved region of 12S rRNA has been found. This method can also be of great help in studying molecular evolution mechanisms.     

The primary structure of mouse saposin.

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.     

Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor.

A human genomic DNA library was screened by using conditions of reduced stringency with a bovine cDNA probe coding for the kringle domains in prothrombin in order to isolate the human prothrombin gene. Twelve positives were identified, three of which coded for prothrombin (Degen & Davie, 1987). Phage L5 was characterized in more detail because of its strong hybridization to the cDNA probe and its unique restriction map compared to the gene coding for human prothrombin. The gene in L5 was sequenced and found to code for a kringle-containing protein. A human liver cDNA library was screened by using a genomic probe from the gene in L5. cDNAs were isolated that contained sequence identical with regions in the gene in L5. Comparison of the cDNA with the gene indicated that the gene in L5 was composed of 18 exons separated by 17 intervening sequences and is 4690 bp in length. Exons ranged in size from 36 to 242 bp in length while intervening sequences ranged from 77 to 697 bp in length. The putative protein encoded by the gene in L5 contains four kringle domains followed by a serine protease-like domain. This domain structure is identical with that found in hepatocyte growth factor (HGF), although the two proteins are only about 50% identical. On the basis of the similarity of the protein encoded by L5 and HGF, we propose that the putative L5 protein be tentatively called HGF-like protein until a function is identified. The DNA sequence of the gene and cDNA and its translated amino acid sequence were compared against GenBank and NBRF databases. Sequences homologous to DNF15S1 and DNF15S2, human DNF15S2 lung mRNA, and rat acyl-peptide hydrolase were identified in exon 17 to the 3' end of the characterized sequence for the gene. From our results, it is apparent that the gene coding for human HGF-like protein is located at the DNF15S2 locus on human chromosome 3 (3p21). The gene for acyl-peptide hydrolase is 444 bp downstream of the gene coding for HGF-like protein, but on the complementary strand. The DNF15S2 locus has been proposed to code for one or more tumor suppressor genes since this locus is deleted in DNA from small cell lung carcinoma, other lung cancers, renal cell carcinoma, and von Hippel-Lindau syndrome.     

ApoE: The role of conserved residues in defining function

The amino acid sequences of apolipoprotein E (apoE) from 63 different mammalian species have been downloaded from the protein database. The sequences were compared to human apoE4 to determine conserved and non‐conserved sequences of amino acids. ApoE4 is the major risk factor for the development of late onset Alzheimer's disease while apoE3, which differs from apoE4 by a single amino acid change at position 112, poses little or no risk for the development of this disease. Thus, the two proteins appear to be structurally and functionally different. Seven highly conserved regions, representing approximately 47 amino acids (of 299) have been found. These regions are distributed throughout the protein and reflect ligand binding sites as well as regions proposed to be involved in the propagation of the cysteine–arginine change at position 112 to distant regions of the protein in the N‐ and C‐terminal domains. Highly non‐conserved regions are at the N‐ and C‐terminal ends of the apoE protein.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6

Ribosomal protein (rp) S6 is the major substrate of protein kinases in eukaryotic ribosomes. To facilitate the identification of cloned cDNAs for human rpS6, we used published amino acid (aa) sequence data for rat liver rpS6 and yeast (Saccharomyces carlsbergensis) rpS10 to design mixed oligodeoxynucleotide probes. Screening of several human cDNA libraries with these probes permitted the isolation of plasmids which encompass the entire coding sequence of rpS6 (249 aa residues), 27 bp of the 5'-untranslated leader and all 39 bp of the 3'-untranslated region. A comparison of the predicted human rpS6 amino acid sequence and the yeast rpS10 amino acid sequence shows highly conserved areas separated by regions of divergence.