Greatest Homology Research Articles

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.

Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase

Methods are described for preparing and structurally analyzing two enzymes involved in the formation of dTMP, deoxycytidylate deaminase and thymidylate synthase. In the latter case, it has been possible through the use of recombinant DNA techniques with an amplification plasmid to obtain sufficient amounts of the E. coli and T4-phage synthases to complete the entire sequence of both enzymes by employing a combination of protein and DNA sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89–139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The DNA and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence of T2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. From the information on the enzyme's primary sequence, it should be possible to define the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for β-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The β-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of β-galactosidase is inducible by isopropyl-β- d-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for β-galactosidase than there is between the respective repressor genes.

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes

Hybridoma-derived monoclonal anti- p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20–70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, κ and IgG2b, κ. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial V H amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarity determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the V H regions from 14 CRI + and 8 CRI − antibodies as well as the V L regions from 11 CRI + and 8 CRI − monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI + and CRI −) which have identical sequences for V H or V L. This suggests that there exist a minimum of 4 germ line genes coding for CRI + v H, CRI + V L, CRI − V H and CRI − V L respectively. In addition, CRI + V L has always been found in association with a CRI + V H.

Direct evidence of homology between human DC-1 antigen and murine I-A molecules.

The nature of the interactions between cells of the immune system has been shown to be directly related to a group of antigens present on the surface of B lymphocytes. These antigens are under genetic control of the HLA-D/DR region of the human major histocompatibility complex (MHC) located on chromosome 6. DR antigens (the products of the HLA-DR locus within this region), have been defined by serological studies. Recently another antigen, DC-1, has been serologically defined. In the mouse, the I region, analogous to the HLA-D/DR region in man, has been subdivided into at least five subregions (A, B, J, E and C) and specific functions and cell populations associated with each of the subregions. Although in man the HLA-D/DR region cannot at present be subdivided, limited NH2-terminal amino acid sequence data have shown that the DR molecule is homologous to the I-E molecule of the mouse. Furthermore, evidence that DC-1 is not homologous to I-E (and might therefore be homologous to I-A) has been reported. We now report that, using immunoadsorbents prepared with monoclonal antibodies specific for DR or DC-1 antigens, these two human alloantigens were purified from a human lymphoblastoid cell line (LB) and the chains were separated. The NH2-terminal amino acid sequence of the heavy chain of DC-1 antigen shows great structural homology with the murine I-A molecules, thus providing direct evidence of homology of DC-1 to murine I-A.

Analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.

Characterization of two forms of glucoamylase from aspergillus niger

Aspergillus niger glucoamylases GI and GII (E.C. 3.2.1.3) were isolated from a commercial enzyme preparation by ammonium sulfate precipitation followed by DEAE-cellulose ion exchange chromatography. Both enzymes consist of a single glycosylated polypeptide chain. The molecular weights of GI and GII were determined by sedimentation equilibrium ultracentrifugation to 52,000 and 46,000, respectively, and by molecular sieving to 65,000 and 55,000. The amino acid compositions of GI and GII were very similar. Furthermore, the N-terminal amino acid sequence of the intact GI and GII as well as of their cyanogen fragments were identical, suggesting great homology in the primary structure of the two forms. In addition the digests of GI and GII produced respectively by Armillaria mellea protease, Staphylococcus aureus V8 protease, and submaxillary protease were analyzed by high pressure gel permeation chromatography. The elution profiles were also consistent with GI and GII having similar polypeptide chains. However, digestion with carboxypeptidase Y showed different C-terminal residues of the two forms.

The 5.8S RNA gene sequence and the ribosomal repeat of Schizosaccharomyces pombe.

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.

A tentative elucidation of the sequence of human triosephosphate isomerase by homology peptide mapping

By utilizing homologous peptide mapping and amino acid analysis of recovered peptides, it has been possible to infer much of the sequence of human triosephosphate isomerase. This has been achieved with less than 5 mg of the human enzyme. The enzyme shows a great sequence homology with the rabbit enzyme. The methods used in this study can provide a rapid procedure for gaining structural information on human enzymes available only in microgram or milligram quantities.

The nucleotide sequence of the replication origin of plasmid NTP1.

The sequence of the DNA of the origin region of NTP1 has been obtained. Analysis of the sequence indicates that: (1) there is great sequence homology in the DNA upstream from the origin in NTP1, ColE1, CLODF13, PBR345 AND PBR322; (2) only seven base pairs of NTP1 are identical with the sequence downstream from the origin in ColE1, although some homology exists for 140 bases downstream; (3) two ten base pair direct repeats are present in NTP1 which are also conserved in all four plasmids named above; (4) probably no polypeptide greater than fifteen amino acids in length is encoded by the NTP1 origin region, since no single open reading frame is conserved in all five plasmids.

Distribution of sequences homologous to the DNA of herpes simplex virus, types 1 and 2, in the genome of pseudorabies virus.

The distribution of related sequences between the genomes of pseudorabies virus (PRV) and herpes simplex virus, types 1 (HSV-1) and 2 (HSV-2), was determined. Approximately 7% of the sequences in PRV are shared by HSV-1 and HSV-2 DNAs. By means of the Southern blot technique, it was found that the homologous sequences are not sequestered in one region but are distributed throughout the PRV genome. HSV-1 and HSV-2 have the greatest homology with the long unique region of PRV DNA and the least with the inverted repeat regions of the molecule. HSV-1 DNA also has few sequences homologous to the short unique region of the PRV genome; HSV-2 DNA hybridizes well to this region. There was no homology of HSV-1 or HSV-2 DNAs with the extreme ends of the inverted repeat regions of PRV DNA.

Isolation and characterization of triosephosphate isomerase isozymes from human placenta

Two isozymes of triosephosphate isomerase have been isolated to homogeneity from human placenta. Triosephosphate isomerase B (the isozyme found in most differentiated human tissues) and triosephosphate isomerase A (a major form found in lymphoblasts, flbroblasts, and certain neoplastic tissues) were compared in terms of their chemical, physical, and biological properties. The two isozymes exhibit essentially identical catalytic properties, but the A isozyme is much more labile. A series of electrophoretic and isoelectric focusing studies indicates the unique properties of each isozyme. Subunit size, amino acid compositions are essentially identical and suggest a strong structural homology with the enzyme from rabbit. Triosephosphate isomerases A and B both contain carboxyl terminal glutamines and amino terminal alanines. Thus, the two isozymes do not appear to be the result of protease modification. The positions of the two methionine and five cysteine residues appear to be identical as well as the new amino terminal residues exposed upon cleavage with CNBr. Tryptic fingerprints while revealing great sequence homology between the two isozymes indicate several unique peptides. The isozymes, therefore, do not seem to represent simple posttranscriptional modifications such as phosphorylation. The far ultraviolet circular dichroic spectra indicate a modest but significant difference in the secondary structure of the two isozymes. The fractional α-helix and β-pleated sheet of triosephosphate isomerase A are within experimental error of that determined from crystallographic analysis of the chicken enzyme, while the B isozyme contains significantly more α-helix. The interpretation most consistent with all of the data is that the two isozymes are the result of two independent cistrons.

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles. Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom. There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca 2+ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution. The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.

The relationship of degradative and resistance plasmids of Pseudomonas belonging to the same incompatibility group.

THE fluorescent pseudomonads include Pseudomonas which can cause serious infections in man, and soil bacteria such as P. putida. Resistance (R) plasmids have been found in strains of both species and degradative (D) plasmids have been demonstrated in strains of the latter species. Where such plasmids can be transferred by conjugation to a common host strain, it has been possible to establish whether they can stably co-exist. Inability to co-exist (incompatibility) is the basis of their classification1,2. It has recently been suggested that both the IncP-2 and IncP-9 groups contain both R and D plasmids2,3. In addition the IncP-2-plasmids are both very large4. Among R plasmids of enterobacteria, incompatibility is generally an indication of evolutionary relationship in that the DNAs of incompatible plasmids show greater homology with each other than with those of compatible plasmids5. However, cases of incompatible plasmids lacking significant homology have also been reported5,6. We have examined the extent to which two R plasmids from P. aeruginosa and two D plasmids, one from P. putida and the other from P. aeruginosa (all assigned to the IncP-9 group3,15,16) share common sequences. The results suggest that plasmids of the same incompatibility group are not necessarily closely related.

Comparative structural analysis of myosin after limited tryptic hydrolysis by use of two-dimensional gel electrophoresis

Polypeptides obtained by limited tryptic digestion of several myosins have been analyzed by a two-dimensional electrophoresis technique. The different maps thus obtained exhibit some common and distinct features specific of the myosins studied. Myosins from rabbit, fast and slow muscle and cardiac tissue, as well as beef cardiac myosin, have been compared. The polypeptides obtained vary in molecular weight from 120 000 60 15 000. The light chains LC1 and LC2, have disappeared. A peptide which comigrates with fast type LC3 is however found. The fast myosin hydrolyzate is very different from that obtained by the hydrolysis of slow and cardiac myosin. Numerous peptides are common to cardiac and slow myosins. However a few peptides are specific of the two myosin types. In the hydrolyzate from fetal calf myosin, some of the typical slow, fast and cardiac peptides can be found. However several apparently unique fetal peptides are also present. The comparison of the fetal calf tissue myosin hydrolyzate and that of [35S] methionine labeled myosin from myotubes in cultures shows qualitatively a very great homology. Thus the protein synthesized by cultured cells seems to be very similar to or the same as that of the embryonic tissue.

Horsetail (Equisetum telmateia) ferredoxins I and II. Amino acid sequences.

Two ferredoxins were isolated from horsetail (Equisetum telmateia) and their amino acid sequences were determined by use of a sequence analyzer in combination with carboxypeptidase digestion and manual Edman degradation of tryptic peptides of carboxymethyl-ferredoxins. Ferredoxins I and II each had only four cysteine residues in a total of 95 and 93 residues, respectively. The amino-terminal residues of both ferredoxins were heterogeneous, but alanine was concluded to be their genuine terminal residue. The comparison of these isozymelike molecules showed 29 differences in amino acid residues with three inverted replacements. One gap was inserted in ferredoxin II at position 32 to align the ferredoxins with greatest homology. Despite the many differences in amino acid residues there was no difference in net charges of the two ferredoxins.

RNAs of influenza virus recombinants derived from parents of known virulence for man.

Extensive use of recombinants made from A/PR/8/34 (H0N1) and wild, virulent H3N2 viruses as live influenza vaccines has provided a number of viruses of defined virulence for man. Clinical symptoms produced by these strains have ranged from febrile influenza to local coryzal symptoms or nil. A study was therefore made of the extent to which the PR8 genome had been incorporated into that of a number of the recombinants. By RNA--RNA hybridization it seemed that recombinants which had 55 per cent of greater homology with the PR8 parent were likely to conform an acceptable standard of attenuation. Those with lesser homology were frequently, but not always, clinically virulent. The technique seemed potentially useful, therefore, for screening PR8 live vaccine recombinants in vitro before giving them to volunteers.

Isolation and characterization of the three light chains from carp white muscle myosin.

Myosin from carp white muscle contains two mol of "DTNB" light chain (mol. wt 17 500 daltons) and two mol of "alkali" light chains (mol. wt 25 000 and 16 400 daltons). The three light chains have been isolated in pure state and characterized by electrofocusing, ultraviolet absorption, amino acid analysis and tryptic peptide mapping. Our results show a great homology between the two carp alkali light chains whereas LC2 seems chemically more different. But the homology of LC1 and LC3 is not so extensive as in the case of the higher vertebrate myosin.

Partial amino acid sequences of the heavy chains of human HLA histocompatibility antigens

HLA antigens are composed of two polypeptide chains, the heavy chain carrying the alloantigenic determinants and the light chain identified as β 2-microglobulin. By the use of microsequencing techniques, the amino terminal sequences of two HLA heavy chain preparations carrying different HLA allospecificities (A2 and B7, 14) have been determined through position 22. With the exception of one position, the two are identical through 22 residues. There is also a considerable degree of homology with mouse H-2 antigens. These data support the hypothesis that the HLA-A and B gene products have evolved by gene duplication from a common ancestral gene and that these gene products have great homology in the primary structure.

Structure of HLA antigens: amino-acid and carbohydrate compositions and NH2-terminal sequences of four antigen preparations.

Amino-acid and carbohydrate compositions in addition to limited NH2-terminal sequence data of four preparations of HLA antigens, including products of both of the major histocompatibility loci (HLA-A and HLA-B), show that the antigens have great homology in the primary structure. A striking observation is that HLA antigens also have a considerable structure homology with mouse H-2 antigens. HLA antigens appear to have one carbohydrate side chain which has the same composition in all four cases. The possible relation of the structure of the heavy chain of HLA antigens to immunoglobulins is discussed, based on the limited data which are available so far.