DNA rearrangements generating artificial promoters.

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.

[Similar organization of beta,beta'-subunit RNA-polymerase genes and adjacent ribosomal protein genes in Enterobacteriaceae and Pseudomonas putida]. / Skodnaia organizatsiia genov beta,beta'-sub''edinits RNK-polimerazy i prilezhashchikh genov ribosomnykh belkov u bakterii kishechnoi gruppy i Pseudomonas putida.

The genes coding for the RNA-polymerase beta,beta'-subunits and adjacent ribosomal protein genes in Escherichia coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, Proteus mirabilis and Pseudomonas putida are compared by the Southern hybridization procedure. In all the species studied close clustering of the genes rplKAJL and rpoBC is demonstrated. Preliminary physical maps for these genes in S. typhimurium, S. flexneri, S. marcescens and P. mirabilis are proposed. Rifampicin is shown to stimulate the beta,beta'-subunit synthesis in all the species studied, suggesting the existence of attenuators localized in front of the rpoBC genes. The similar arrangement of the genes rplKAJLrpoBC in a number of bacterial species is proposed to be due to common mechanisms of their coordinate expression.

[Cloning of the promoter of the RNA polymerase operon located before the structural gene of the beta-subunit]. / Klonirovanie promotora RNK-polimeraznogo operona, raspolozhennogo pered strukturnym genom beta-sub"edinitsy.

An EcoRI-fragment of the rpoBC operon carrying the end of the rplL gene, the intercistron region and the beginning of the rpoB gene cloned on the pBRH4 plasmid makes it tetracyclin-resistant, i. e. possesses the properties of a promoter. There is no appreciable difference in the degree of antibiotic resistance of recombinant plasmids carrying the main PJ promoter and the additional promoter.

[Effect of rifampicin on the synthesis of bacterial RNA polymerase mRNA by means of hybrid plasmids]. / Issledovanie vliianiia rifampitsina na skorost' sinteza mRNK bakterial'noi RNK-polimerazy s pomoshch'iu gibridnykh plazmid.

We studied the rate of synthesis of beta- and beta'-subunits of DNA-dependent RNA polymerase and the rate of beta-polypeptide mRNA synthesis in rifampicin-treated bacteria. The antibiotic doses used did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It is found that low doses of rifampicin cause an absolute and differential increase in the rate of synthesis of the specific mRNA for the beta-subunit, suggesting a stimulation of the corresponding gene transcription. However the absolute transcription stimulation does not fully correlate with the relative acceleration of beta-mRNA and the corresponding polypeptide synthesis. The stimulating effect of rifampicin on the beta-polypeptide synthesis was demonstrated also in a coupled system of transcription and translation directed by lambda rifd 47 DNA. The possible mechanisms of the rifampicin action are discussed.

[Nucleotide sequence of the barley C-hordein gene]. / Nukleotidnaia posledovatel'nost' gena C-gordeina iachmenia.

The nucleotide sequence of barley C-hordein gene lambda CH4 and its flanking regions of 2820 bp length was determined. The gene contains no introns and codes for 310 amino acid long polypeptide. The 94% of the deduced amino acid sequence of the mature protein (291 amino acids) is made up of a repeating octapeptide motiff, PQQPEPQQ, which is repeated throughout the peptide chain between a unique 12 amino acid long NH2 terminal and a unique 6 amino acid long COOH-terminal end. In the 5' non-coding region there are TATA-, AGGA-, CAAT-and "endosperm" boxes. The 3' non-coding region has two polyadenylation signals. Compared with the published C-hordein sequences, our gene contains a number of insertions, deletions and substitutions. In the 3'-untranslated region there are two insertions 157 and 23 bp long. For the longer insertion no significant homology was found in the Gene Bank datebase. This insertion is the largest known rearrangement in the otherwise highly conservative surroundings of barley storage protein genes.

[Structural organization of C-hordein genes from barley]. / Strukturnaia organizatsiia genov C-gordeinov iachmenia.

Three nucleotide sequences from the barley genome, containing the C-hordein genes lambda CH1, lambda CH3, and lambda CH5, were cloned. They were shown to have different physical maps, and the structural organization of homologous sequences was found to be highly conservative. The surrounding sequences of C-hordein genes contain insertions specific in length and copy number. A Hind III fragment of about 3000 bp in size of the lambda CH5 clone contains a highly repeated nucleotide sequence. The lambda CH3 clone contains two "head-to-head" repeated C-hordein genes. This is the first evidence that hordein genes may be located in the immediate vicinity of each other in a 17,800-bp region. A 919-bp nucleotide sequence of one of the C-hordein genes from this clone, pCHOR3, was determined. An open reading frame of 363 bp codes for a potential polypeptide consisting of 121 amino acids. The main part of the mature polypeptide consists of repeated octapeptide Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motifs. The coding region has no introns. The 5' untranslated region contains regulatory loci specific for prolamin genes: the -300 element and the CAAT, AGGA, and TATA boxes. Significant differences in the lengths of the coding regions of cloned genes were observed: 363 and 1000 bp in the lambda CH3 clone, 500 bp in the lambda CH1 clone, and 600 bp in the lambda CH5 clone, which may be a molecular cause of C-hordein size polymorphism.

[Bacillus subtilis gene rec223: molecular cloning and proposed function of its protein product]. / Gen rec223 Bacillus subtilis: molekuliarnoe klonirovanie i predpolagaemye funktsii ego belkovogo produkta.

Chromosomal DNA fragment which complemented rec223 mutation of Bacillus subtilis was cloned. Introduction of one copy of the cloned gene into the cells of the rec mutant restored both normal activity for DNA damages repair after mitomycin C action and recombination proficiency. Using multicopy vector led to no formation of recombinants, which was probably connected with overproduction of rec223 gene protein product in Bacillus subtilis cells.

[Nucleotide sequence of the B1-hordein gene of barley Hordeum vulgare L]. / Nukleotidnaia posledovatel'nost' gena B1-gordeina iachmenia Hordeum vulgare L.

The gene encoding B1 hordein of Hordeum vulgare (cv. Donetsky 4) was cloned and entirely sequenced. It contains no introns and codes for of 293 amino acids long polypeptide with molecular weight 33418. Our clone differs from the previously sequenced B1 hordein genes in some positions within the coding region (there are 4 nucleotide changes and a 12 bp deletion, as compared with the pB11 cDNA clone, and 5 nucleotide changes, as compared with the pBHP184 genomic clone). These changes result in polymorphism of amino acid sequences at 5 positions. 5'-flanking region contains putative regulatory and promoter sequences and differs from that of the pBHP184 clone in 3 positions.

Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes.

Partial submergence or treatment with either ethylene or gibberellin (GA) promotes rapid internodal growth in deepwater rice (Oryza sativa L.). Earlier work has shown that GA is the immediate hormonal signal for this growth response, which involves induction of the cell cycle at the G2/M phase transition and subsequent enhancement in the rate of DNA synthesis. In all eukaryotes, onset of mitosis is regulated by the p34cdc2/CDC28 protein kinase, whose activity is assayed by in vitro phosphorylation of histone H1. It was found that GA enhanced the activity of p34cdc2/CDC28-like histone H1 kinase in the intercalary meristem of rice internodes. The enzyme activity showed a sharp peak that correlated with a decrease in the population of cells in the G2 phase during the first 4 h of GA treatment but not with changes in DNA synthesis. The level of histone H1 kinase activity increased again when cell division activity in the intercalary meristem is known to be high. The expression of two cdc2 homologs was examined. The mRNA level of one of these, cdc2Os-2, was increased after 1 h of GA treatment, whereas the mRNA level of the other, cdc2Os-1, was not affected. Two cDNAs, cycOs1 and cycOs2, which show high homology to cyclin cDNAs, were cloned from rice. They share 75.1% sequence identity at the amino acid level, and both of them are encoded by mRNAs of 1.6 kb. Expression of the two corresponding cyclin genes was enhanced by GA, and the time course of the induction was compatible with a role for both cyclins in regulating the G2/M phase transition. The cyclins were expressed in the intercalary meristem and the elongation zone of the internode, but the GA-induced increase in transcript levels was restricted to the meristem only. The results support the hypothesis that induction of mitosis by GA is brought about by increased p34cdc2/CDC28 protein kinase activity, which may be the result of transcriptional activation of the cdc2Os-2, cycOs1 and cycOs2 genes.

The effect of rifampicin upon the transcription of RNA polymerase beta-gene in Escherichia coli.

We studied the rate of synthesis of beta-and beta'-subunits of DNA-dependent RNA polymerase and the rate of beta-polypeptide mRNA synthesis in rifampicin-treated bacteria. The chosen antibiotic doses did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It was found that low doses of rifampicin cause an absolute and a relative increase in the rate of synthesis of the specific mRNA for the beta-subunit, suggesting a stimulation of the corresponding gene transcription and excluding the possibility of a less pronounced inhibition of the rpoB gene expression compared to that of most other genes. However the relative acceleration of transcription is substantially higher than the absolute one. The stimulating effect of rifampicin on the beta-polypeptide synthesis is also demonstrated in a coupled system of transcription and translation directed by lambda rifd47 DNA. The possible mechanisms of the rifampicin action are discussed.