Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.

[The genetic transformation of Lycopersicon peruvianum var. dentatum by using Agrobacterium tumefaciens carrying a plasmid with the human beta-interferon gene]. / Geneticheskaia transformatsiia Lycopersicon peruvianum var. dentatum s pomoshch'iu Agrobacterium tumefaciens, nesushchei plazmidu s genom beta-interferona cheloveka.

Wild tomato plants of Lycopersicon peruvianum var. dentatum were transformed with pG11 plasmid having human beta-interferon (hIFN-beta) and NPTII genes. Transformation was demonstrated by polymerase chain reaction (PCR) and ELISA assay. Expression of hIFN-beta gene was identified by Western blot analysis. Transgenic plants produced seeds and F1 generation inherited integrated traits.

[Primary structure of the coding part of the gene for human pancreatic ribonuclease and its chromosomal location]. / Pervichnaia struktura kodiruiushchei chasti gena pankreaticheskoi ribonukleazy cheloveka i ego lokalizatsiia na khromosomakh.

On the basis of the known primary structure of the gene for murine pancreatic ribonuclease, two deoxyoligonucleotides were selected as primers for amplification of human pancreatic ribonuclease gene. The PCR amplified DNA fragment was subsequently cloned, and its nucleotide sequence was determined. Pancreatic ribonuclease gene was localized on human chromosome 14.

[Tyrosine hydroxylase activity and expression of its gene in mice with contrasting capacity to dominate in social stress]. / Aktivnost' tirozingidroksilazy i ékspressiia ee gena u kontrastnykh po sposobnosti k dominirovaniiu myshei pri sotsial'nom stresse.

Changes of tyrosine hydroxylase (TH) activity and level of mRNA of TH gene in PT and CBA/Lac mouse strains, which are contrast by ability to dominate in heterogenous populations, were investigated. It was established, that the activity of TH both in dominate PT and subordinate CBA/Lac mice in hypothalamus, hippocampus and brain stem elevated in one hour after forming of micropopulations. But the appearance of this increase was different: activation of TH in hypothalamus and brain stem of PT mice was stronger then one in CBA/Lac mice. Moreover, the beginning of the reaction in brain stem of PT mice was earlier then that of CBA/Lac mice. MRNA level of TH gene in hypothalamus and brain stem in one hour was elevated only in PT mice for 50% and 200%, respectively. No changing in expression TH gene was found in hippocampus. In conclusion, it was suggested that the activation of catecholamine biosynthesis under social stress in hypothalamus and brain stem of male mice was due to the TH activation and increase of its gene expression.

B2-like repetitive sequence from the X chromosome of the American mink (Mustela vison).

In analysis of the repeats from the mink X Chromosome (Chr), we have identified a B2-like repetitive sequence of 195 base pairs (bp) flanked by short direct repeats of 14 bp. It contains regions homologous to the split intragenic RNA polymerase III promoter and a 3' A-rich region followed by an oligo(dA) sequence. A feature of the repeat is the presence of a perfect polypyrimidine tract 22 bp in length absent from the known Alu- and Alu-like sequences. Alignment of the mink B2-like sequence and mouse B2-consensus sequence allowed us to estimate their similarity as 55%. The repeat is present in 1-2 x 10(5) copies per mink genome and 2-4 x 10(3) copies per X Chr. In situ hybridization analysis demonstrated a similar distribution pattern of the B2-like repeat along the length of all the mink chromosomes including the X. We also observed the presence of mink B2-like hybridizable sequence in the genomes of other Carnivora species.

Primary structure of the gene coding for the haemagglutinin of influenza virus A/Leningrad/385/80(H3N2): detection of a point mutation responsible for the antigenic drift.

Primary structure of the gene coding for haemagglutinin (HA-gene) of influenza virus A/Leningrad/385/80(H2N2) isolated during the epidemics of influenza in Leningrad in 1980 was determined. The close relationship of HA gene of this virus to the corresponding gene of the virus A/Bangkok/1/79(H3N2) was confirmed. It was shown that a single mutation in an antigenic site (the change from isoleucine to leucine at position 51 of HA1 gene) caused an antigenic drift. One silent mutation was detected (nucleotide 428 of HA1 gene) which points at the relatedness of strains A/Leningrad/385/80 with A/Bangkok/2/79 and with other more recent strains. These data allowed to determine the position of the strain A/Leningrad/385/80 HA gene regarding to the evolutionary relationships of HA genes of influenza A (H3N2 subtype) viruses. The branch leading to the above-mentioned strain is supposed to start from a point common for strains isolated following A/Bangkok/1/79. The mutations of HA genes presented in this subgroup were analysed supporting the notion on limited evolutionary potential of the subtype H3N2 influenza viruses.

[An improved chemico-enzymatic method of synthesizing long DNA segments]. / Usovershenstvovannyi khimiko-fermentativnyi metod sinteza protiazhennykh fragmentov DNK.

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.

[Plasmid vector pSSC1 for cloning synthetic polynucleotides and their regeneration with a unique nucleotide sequence]. / Plazmidnyi vektor pSSC1 dlia klonirovaniia sinteticheskikh polinukleotidov i ikh regeneratsii s unikal'noi nukleotidnoi posledovatel'nost'iu.

For subcloning separate synthetic gene fragments, a plasmid vector pSSC1 was constructed by inserting a synthetic polylinker into plasmid pBR 327 at the EcoRI-PstI sites. There are two FokI and HgaI sites at the ends of this polylinker in the opposite orientation, with the EcoRI and PstI sites between them. DNA fragments cloned at the EcoRI and PstI sites can be regenerated by either FokI or HgaI, the EcoRI and PstI sites being deleted from the cloned sequences. Such fragments have unique cohesive ends that allows their directed ligation into longer DNA (genes).

[Determination of the primary structure of oligodeoxyribonucleotides containing P-S-C(5') bonds]. / Opredelenie pervichnoi struktury analogov oligodezoksiribonukleotidovs P-S-C(5')-sviaziami.

Analogues of oligodeoxynucleotides with P-S-C(5') bonds, which, due to their unusual substrate properties, may find interesting applications in molecular biology, can not be structurally analysed by the Maxam-Gilbert or Sanger (fingerprinting) methods. We, therefore, devised a modification of the fingerprinting technique making possible the sequence determination of these analogues.

[Cloning of single-stranded synthetic DNA]. / Klonirovanie odnotsepochechnykh sinteticheskikh DNK.

The method for cloning a single-stranded synthetic DNA with the short complementary oligonucleotides, that form corresponding restriction sites, is proposed. The potency of the method is demonstrated by cloning a single-stranded polynucleotide A (93 nucleotide residues (n. r.] in plasmid vector pBR327. The polynucleotide A includes a leader structure of the human fibroblast interferon gene. Oligonucleotides (IV) (20 n. r.) and (VI) (16 n. r.) were taken as strengthening complements and to create the sticky ends for the restrictases HindIII and EcoRI. 72% of the obtained clones appeared to be hybrid. Four hybrid clones were analyzed, and three of them carried the desirable insertion. The primary structures of these insertions are confirmed by sequencing.

Some substrate properties of analogues of oligothymidylates with p-s-C5' bonds.

The action of T4 polynucleotide kinase, T4 DNA polymerase, E. coli DNA polymerase I, snake venom phosphodiesterase (VPDE) and S1 nuclease on analogues of oligothymidilates with p-s-C5' bonds and the ability of these analogues to prime the replication of poly (dA) by T4 DNA polymerase were studied. These analogues were shown to be substrates for all these enzymes. Substitution of these analogues for corresponding oligothymidilates in the reaction mixtures resulted in drop in rates of enzymic reactions. This drop in reactions rates was not significant when these oligonucleotides were phosphorylated with T4 polynucleotide kinase or used as a primers, however in comparison with oligothymidilates these analogues were found to be considerably more resistant to nucleolytic hydrolysis. Some possible applications of these analogues are discussed.