Stretches Of Homology Research Articles

The primary structure of protein L2 from the Escherichia coli ribosome

The complete primary structure of protein L2 which is the largest protein component of the E. coli 50 S subunit, has been established. A combination of enzymatic and chemical cleavages has been employed to isolate peptides, which were sequenced by the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205–214]. The sequence determined shows protein L2 to consist of 272 amino acid residues with M r = 29730. Secondary structure predictions were made based on the primary structure. Further, sequence regions homologous to other ribosomal proteins are presented. These results suggest protein L2, which binds specifically to the 23 S RNA, to show homologous sequence stretches to the other RNA-binding proteins.

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. II. Unstable association of heterologous DNA with the recipient chromosome.

In CsCl density gradients of lysates from competent Bacillus subtilis cells, which had been exposed to heterologous bacterial DNA, very little donor-recipient complex (DRC) formation could be detected. The present study demonstrates that photocrosslinking of such lysates by irradiation with long-wave UV light in the presence of 4,5',8-trimethylpsoralen results in a dramatic increase in the amount of heterologous DRC. This phenomenon may be interpreted as the stabilization of a pre-existing weak association between entered heterologous donor DNA and one recipient strand in unpaired regions of the chromosome. When a recombination-deficient mutant is used, the amount of stabilizable heterologous DRC is reduced to the same extent as the specific transforming activity of homologous DNA. Although the amount of stabilizable complex is related to the degree of homology between donor and recipient DNA, this relation is not a quantitative one. Probably the association is caused by very short regions of base pairing between the donor and recipient moieties in the complex. Heating of a lysate at 70 degrees prior to photocrosslinking prevents stabilization, apparently because the regions of base pairing are rapidly melted out. The results described in this paper can be best interpreted as the fixation of a process in which entered donor DNA in competent cells tries to find homologous stretches in the recipient chromosome.

Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus.

Twelve cyanogen bromide fragments (CB1-12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1-4) constitute residues 1-289 in fibronectin with CB4 overlapping the N-terminal 29-kDa plasmic fragment to the second plasmic fragment, of 170-kDa in fibronectin. Fragments CB 5-9 are all contained within a 45-kDa gelatin-binding region, which is N-terminal in the 170-kDa fragment. The sequence of two of these five fragments in the 45-kDa fragment (CB7-8) contains two mutually homologous stretches with 57% sequence identity. Another two fragments (CB10-11) are derived from the heparin-binding region of the 170-kDa fragment. CB12 constitutes the C-terminal 13-residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C-terminal sequence: -Arg-Glu-Asp-Ser(P)-Arg-Glu.

Human IgG Allotypes Co-Occurring in More than One IgG Subclass

Inheritance of an excess of immunoglobulin allotypes in one haplotype was encountered which could not be explained by the assumption of a duplicated locus. The surplus of allotypes was related to markers on the CH3 domain of y3 chains. Two such cases were investigated extensively. The IgG3 molecules were isolated by gel filtration and by absorption on protein A. Only the usual combination of allotypes appeared to be present on the IgG3 molecules. The supernumerary markers were found in one case on IgG2 molecules and in the other case on IgG 1 molecules. This followed from investigations of eluates after separation of the subclasses by immune absorptions. A hypothesis was proposed to explain these events by mutation of a particular position of an otherwise homologous stretch of y-subclass DNA.

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.

The origins of replication of the yeast mitochondrial genome and the phenomenon of suppressivity.

The ‘petite colonie’ mutation of Saccharomyces cerevisiae1,2 is characterized by an irreversible loss of respiration and by an extraordinarily high spontaneous mutation rate3,4. Crosses of wild-type cells with petite mutants exhibit a non-mendelian segregation of the mutation, yielding either wild-type progeny only1,2, or both wild-type and petite mutants in proportions essentially dependent on the particular petite used3. In the first case, the petites entering the cross are called neutral, in the second one suppressive3. While the molecular basis of the spontaneous petite mutation is now understood4–12, suppressivity has remained an elusive phenomenon for the past 25 yr. We report here that the mitochondrial genome of most spontaneous petites (which is exclusively made up by the tandem repetition of a DNA segment excised from the genome of parental wild-type cells5–8) carries at least one of the ori sequences of the parental wild-type genome. These are long homologous DNA stretches showing striking similarities with the origins of replication of mitochondrial DNAs from mammalian cells. The properties (intact or altered primary structure, high or low number) of the ori sequences of petite genomes seem to determine suppressivity—the level of transmission of petite genomes to the progeny of crosses with wild-type cells. These results indicate that ori sequences are indeed origins of DNA replication and that suppressivity depends on the relative replication efficiencies of petite and wild-type genomes.

Isolation and structure of a rat cytochrome c gene.

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.

Isolation and characterization of genomic clones covering the chicken vitellogenin gene.

A series of overlapping recombinant clones, which cover the vitellogenin gene, has been isolated from a phage-lambda linked chicken gene library. The DNA of the overlapping clones spans 28 kb of contiguous DNA sequences in the chicken genome. Electron microscopic analysis of hybrids between vitellogenin mRNA and the genomic clones indicates that the chicken vitellogenin gene has a length of approximately 22 kb, about 3.8 times the size of the mRNA. The mRNA sequence is interrupted by at least 33 intervening sequences (introns). Comparison with the vitellogenin gene A2 from Xenopus laevis (Wahli et al., 1980, Cell 20: 107-117) indicates conservation of the number and length of the exons during evolution. Heteroduplex analysis reveals a short stretch of sequence homology between the genes from chicken and frog.

Evolutionary variants of simian virus 40: Cellular DNA sequences and sequences at recombinant joints of substituted variants

Abstract Cellular DNA and host-viral joints present in the reoombinant genome of an evolutionary variant of simian virus 40 (SV40) have been analyzed at the nucleotide sequence level. Cellular DNA segments are interspersed with viral DNA and are arranged in part as tandem sequence repetitions. Some of the cellular DNA was derived from highly repeated sequences of host cell DNA and corresponds to sequences found in other substituted SV40 variants. Ten recombinant host-viral joints have been identified in the variant genome, six of which show distinct nucleotide sequences on both sides of the joint. In all but one instance, sequences about the joint were rich in clusters of A·T base-pairs. In one case, joint sequences could be compared with both viral and cellular sequences from which they were derived: short interrupted stretches of homology with each parental sequence were noted. Of the remaining five joints (which could be compared only with the SV40 parental sequence), two showed patchy homology with SV40 sequences adjacent to the joint. Our general conclusions are (1) recombination between SV40 and cellular DNA occurs at many different nucleotide sequence positions in both parental DNAs, (2) extensive nucleotide sequence homology is not required for such recombination, (3) discontinuous or patchy homology may play a role in the recombination event, and (4) A·T clusters may increase the probability of recombination at a given site.

The DNA sequence of sea urchin (S. purpuratus) H2A, H2B and H3 histone coding and spacer regions

The DNA sequence of two cloned segments of the histone gene repeat unit of the sea urchin S. purpuratus has been determined. One sequence contains the contiguous H2B and H3 genes and their interdigitated spacer regions; the other comprises the H2A gene and flanking spacer sequences. Analysis of the coding regions reveals a methionine residue within the H2A protein. H2A, which generally lacks this amino acid, contains methionine only in a protein variant which is synthesized in early sea urchin embryogenesis. We thus conclude that the cloned DNA represents a set of genes which is active early in development. Codon selection is markedly skewed and similar for each of the three genes. The DNA sequences are co-linear with known histone protein sequences and — unlike several other eucaryotic genes — do not show any insertions in the coding regions. The spacer regions are relatively AT-rich although GC clusters are scattered throughout. Several short stretches of homology are found in regions both upstream and downstream from the protein coding segments. The conservation of these sequences and their location at analogous sites suggest that they are involved in gene transcription or in mRNA translation. No tandem or dispersed repeats were found, with the exception of the remarkable sequence having the structure ▪ located in the spacer between the H2A and H1 genes.

Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas

The specificity of the immune response elicited in rabbits to purified bovine prothrombin, autoprothrombin III (Factor X), Factor IX, and Protein C was studied. The immunizing proteins were homogeneous on polyacrylamide gel electrophoresis. By immunodiffusion analysis antigenic similarities were not detected between them in spite of known stretches of amino acid sequence homology. Immunoelectrophoresis of bovine plasma and each purified protein gave identical single precipitin lines with the homologous antisera. This experiment established that the antibodies prepared against each one of the purified vitamin K-dependent factors studied reacted with a single component in bovine plasma. Immunologic crossreactions were found between human, bovine, dog, rat and chicken prothrombin, Protein C, Factor X, and Factor IX. They were not species specific. Purified bovine prothrombin has at least four antigenic determinant sites. Incubation of the purified proteins with suitable proteolytic enzymes yielded fragments and intermediate products which were purified and tested for their potential capacity to react with antibodies in the native antisera. In immunoelectrophoresis tests both prothrombin fragments 1 and 2 and the intermediate product prethrombin 1 gave immunoprecipitates which were separately identified by their differing mobilities, while the enzyme portion, thrombin, did not react with antithrombin serum. In addition to prothrombin, bovine plasma contained prethrombin 1, prothrombin fragments 1 and 2, as well as prethrombin 2 and/or thrombin. Autoprothrombin III m (Factor Xβ) and autoprothrombin C (Factor Xa) crossreacted with anti-autoprothrombin III and the active form of Protein C. Gamma globulins of antisera to prothrombin, to autoprothrombin III, to Factor IX, and to Protein C were fractionated by using saturation ammonium sulfate solution and coupled to activated Agarose A-15m beads. The insolubilized antibodies were used to absorb the antigen from the plasma. Each depleted plasma served as test material for a specific clotting factor. The prolonged prothrombin time of the prothrombin depleted plasma was normalized by additions of purified prothrombin, but not by purified prethrombin 1. The prothrombin time of autoprothrombin III depleted plasma was shortened by fresh plasma, purified autoprothrombin III, and by purified autoprothrombin III m (Factor Xβ). In the case of Factor IX depleted plasma, the partial thromboplastin time was used. The depleted bovine plasmas are suitable for assay purposes. They serve the same role as the corresponding deficient plasma in bioassay coagulation tests.

Presynaptic events in meiocytes of Lilium longiflorum and their relation to crossing-over: a preselection hypothesis.

We are proposing a "Preselection Hypothesis" to account for the regulation of crossing-over in eukaryotic organisms. The hypothesis characterized meiosis in terms of three major physiological stages: (1) a presynaptic stage when pairs of homologous DNA stretches are selected so as to become trapped within the synaptinemal complex during synapsis, (2) an alignment of homologous chromosomes and stabilization of paired bivalents via the synaptinemal complex, and (3) a scission and rejoining of DNA stretches leading to the formation of chiasmata and crossovers. The hypothesis centers on the first stage and is based on evidence for the occurrence of significant cytological and biochemical changes prior to synapsis. The major feature of the hypothesis is that crossing-over occurs only in trapped DNA stretches. Thus, potential crossing-over sites, though not crossing-over itself, are determined well before chromosomes pair. Since, to a large degree, crossovers are distributed randomly along the length of each chromosome, the preselection process must result in a random assortment of trapped DNA stretches, the assortment differing from one meiocyte to another.

Adenomatoid hamartoma of the lung in a newborn infant.

<i>short stop (shot)</i> is required for sensory and motor axons to reach their targets in the <i>Drosophila</i> embryo. Growth cones in <i>shot</i> mutants initiate at the normal times, and they appear normal with respect to overall morphology and their abilities to orient and fasciculate. However, sensory axons are unable to extend beyond a short distance from the cell body, and motor axons are unable to reach target muscles. The <i>shot</i> gene encodes novel actin binding proteins that are related to plakins and dystrophin and expressed in axons during development. The longer isoforms identified are predicted to contain an N-terminal actin binding domain, a long central triple helical coiled-coil domain, and a C-terminal domain that contains two EF-hand Ca²⁺ binding motifs and a short stretch of homology to the growth arrest-specific 2 protein. Other isoforms lack all or part of the actin binding domains or are truncated and contain a different C-terminal domain. Only the isoforms containing full-length actin binding domains are detectably expressed in the nervous system.<i>shot</i> is allelic to <i>kakapo</i>, a gene that may function in integrin-mediated adhesion in the wing and embryo. We propose that Shot9s interactions with the actin cytoskeleton allow sensory and motor axons to extend.