Homology Scores Research Articles

Alfalfa cyclins: differential expression during the cell cycle and in plant organs.

Cell division in eukaryotes is mediated by the action of the mitosis promoting factor, which is composed of the CDC2 protein kinase and one of the various mitotic cyclins. We have recently isolated a cdc2 gene from alfalfa. Here, we report the isolation of two cyclin genes, cycMs1 and cycMs2, from alfalfa. The cycMs2 gene shows highest similarity to type B cyclins. In contrast, the predicted amino acid sequence of the cycMs1 gene shows similar homology scores to cyclins of all types (25 to 35%). Both genes are expressed in dividing suspension cultured cells but cease to be expressed when the cells enter stationary phase. In synchronized alfalfa suspension cultured cells, the mRNAs of cycMs1 and cycMs2 show maximal expression in the G2 and M phases. Transcripts of cycMs2 are found only in late G2 and M phase cells, an expression pattern typical for cyclin B genes, whereas cycMs1 appears with the onset of G2. This pattern indicates that alfalfa cycMs1 and cycMs2 belong to different classes of cyclins. In young leaves, expression of both genes is high, whereas in mature leaves no transcripts can be detected, indicating that the two cyclin genes are true cell division markers at the mRNA level. In other organs, a more complex expression pattern of the two cyclin genes was found.

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A] 7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (G NS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67–74). A database search indicated that both the G and G NS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.

Identification and DNA sequence of the large subunit of the capping enzyme from Shope fibroma virus

A 3.6-kb region of the Shope fibroma virus (SFV) BamHI D fragment located in the central region of the viral genome was sequenced. Three open reading frames (ORFs) were identified, D3R, D4L, and D5R. Each of these ORFs have a counterpart organized identically within the HindIII fragment D of the vaccinia virus genome (D1R, D2L, and D3R). Homology scores and assays of viral cores indicate that SFV D3R encodes the large subunit of the SFV mRNA capping enzyme.

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.

Streptomyces griseus Streptomycin Phosphotransferase: Expression of Its Gene in Escherichia coli and Sequence Homology with Other Antibiotic Phosphotransferases and with Eukaryotic Protein Kinases

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a approximately 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of approximately 34.5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence data base was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.

Identification of 21 Genes of Infectious Laryngotracheitis Virus Using Random Sequencing of Genomic DNA

DNA from infectious laryngotracheitis virus (ILTV) was randomly sheared and cloned into the M13 bacteriophage. Clones containing ILTV DNA were sequenced and the predicted amino acid sequences were compared to the known sequences of other herpesviruses using computer analysis. Twenty-one ILTV genes were identified, 20 by comparison to varicella-zoster virus and 19 by comparison to herpes simplex virus type 1; only 12 genes, giving consistently lower homology scores, were found by comparison with the gammaherpesvirus Epstein-Barr virus, indicating that ILTV sequences bear greater similarity to other alpha- than to gammaherpesvirus sequences.

Evidence for a Potential Common T-Cell Epitope Between Human Thyroid Peroxidase and Human Thyroglobulin with Implications for the Pathogenesis of Autoimmune Thyroid Disease

In order to explore the possibility that, in autoimmune thyroid disease, anti-thyroglobulin (Tg) and anti-thyroid peroxidase (TPO) antibodies arise concurrently because they share a common T-cell epitope, we performed a detailed comparative analysis of the cDNA nucleotide sequences corresponding to these two genes. We discovered an 8 amino acid region (Leu-Ser-Glu-Asp-Leu-Leu-Ser- Ile in human TPO) in which there were 6 identical and 2 conserved amino acid residues when compared with human Tg. This remarkably similar region is near the amino-terminus of human TPO (residues 119-126) and the carboxyl-terminus of human Tg (residues 2763-2770). A second feature of interest was that this region of homology conforms to the Rothbard algorithm for a T-cell epitope. Third, probing of the Swiss-protein data bank (10,008 proteins containing 2,952,765 amino acids) with the human TPO region revealed greater homology for human Tg than for any other eukaryotic protein. Two bacterial proteins (E. coli aminopeptidase N and stringent starvation protein) had higher homology scores from human TPO than did human Tg. Our findings provide circumstantial evidence that human TPO and human Tg, and possibly certain bacterial proteins, do indeed share common T-cell epitopes that may play a role in the pathogenesis of autoimmune thyroid disease.

Intermediate filament structure: 3. Analysis of sequence homologies

Homology profiles have been calculated for each chain classification (Types I–V) using all available IF sequence data and detailed analyses of the results have been made. Features of structural relevance which have been noted include the following: (i) The linear distribution of homology scores for the acidic and the basic residues in segments 1B and 2 includes the same periods as those previously observed in the linear distribution of the same residue types in individual chains. This confirms that the spatial distribution of the ionic residues has been maintained during evolution, probably as a consequence of its importance in specifying the aggregation of molecules in IF. (ii) The maintenance of highly conserved sequences at the ends of the rod domain appears crucial to IF structure in general but significant differences in the location of regions of very high (or very low) homology are apparent when one chain type is compared to another. Furthermore, although differences exist between the various types of IF chains, the pronounced homologous features argue that intermediate filaments are a polymorphic family of closely related structures. (iii) The high degree of homology in the L2 link segment in all chain types suggests a highly conserved structure, probably α-helical; a limited degree of homology in all chain types for segment L12 is consistent with a partially conserved structure, possibly β; the highly variable length of link L1, together with the low level of homology present, suggests an irregular conformation. (iv) On the basis of sequence homology the Type I and Type II sequences can each be subdivided. Type Ia and IIa chains constitute those from the ‘hard’ α-keratins and Type Ib and IIb chains constitute those from the ‘soft’ epidermal keratins. (v) The neurofilament chains are confirmed as Type IV chains and not Type III. (vi) The NF-L, NF-M and NF-H neurofilament chains may be clearly distinguished within the Type IV classification on the basis of homology within both the rod domain and the H1 and H2 subdomains. (vii) The extended sequence data base now available has revealed that both the H1 and H2 subdomains differ significantly in extent. For example, the H1 subdomain encompasses the entire N-terminal domain of Type IV chains (both NF-L and NF-M) and the H2 subdomain encompasses the entire C-terminal domain of Type III chains. In contrast the Type I IF chains appear to have negligible H subdomains.

On antisense peptides: The parathyroid hormone as an experimental example and a critical theoretical view

We followed an approach which predicts that translation of two complementary RNA strands into protein generates pairs of “antisense” peptides which bind each other with specific and high affinity (Bost et al. Proc. Natl. Acad. Sci. (1985) 82, 1372). We used human parathormone as an experimental example, and we analysed by computer homologies between antisense peptide sequences and their published receptor sequences. We conclude that there is no experimental indication that parathormone binds to a synthetic peptide, the sequence of which was derived from the antisense RNA sequence. Based on homology scores and antigenicity indexes (Hopp) the analysis shows that the peptide ligand itself, or a random artificial peptide, are as good candidates as the antisense peptide in producing antibodies, presumably recognizing the receptor. We therefore question the general applicability of this approach.