[E.V. Anan'ev's contribution to studies of the centromere and construction of an artificial plant chromosome].

The plant centromere has been described to consist of blocks of repetitive DNA sequences, and self-assembly of an artificial plant chromosome has been achieved using individual cloned elements. E.V. Anan'ev's contribution to these studies is described.

[Hoppel-family of mobile elements of Drosophila melanogaster, flanked by short inverted repeats and having preferential localization in the heterochromatin regions of the genome]. / Hoppel-semeistvo mobil'nykh élementov Drosophila melanogaster, flankirovannoe korotkimi invertirovannymi povtorami i imeiushchee aredpochtitel'nuiu lokalizatsiiu v geterokhromatinovykh oblastiakh genoma.

A mobile element (ME) having 91% homology with Dm1360 (Kholodilov et al., 1987) has been cloned from the Drosophila melanogaster genome and sequenced. The family of ME was designated hoppel. The members of this family are flanked by short inverted repeats likewise P, hobo and HB. The hoppel is hybridized with 10-30 euchromatic sites of polytene chromosomes of different Drosophila stocks. Abundant hybridization with heterochromatic regions of chromosomes-chromocenter, pericentric heterochromatin, the 4 chromosome and telomeres was observed in all stocks of D. melanogaster examined and in D. simulans. At least six genomic variants of ME differing in length of the central part were revealed. Hoppel possesses ARS activity similar to the P element. Two ME hoppel were shown to be arranged as a direct repeat in the recombinant phage.

[Amplification of subtelomeric sequences in "long" telomers of Drosophila melanogaster polytene chromosomes]. / Amplifikatsiia subtelomernykh posledovatel'nostei v "dlinnykh" telomerakh politennykh khromosom Drosophila melanogaster.

Drosophila melanogaster heat-resistant stock T32 is characterized by additional chromatin in the telomeres of the X and 2L chromosomes. Moreover, elevated (32 degrees C) temperature provokes high instability in the telomere morphology, so that sublines can be obtained which have additional chromatin in different chromosomes. Morphological patterns of telomeres in each subline are stable, if flies are kept at 23 degrees C. It was demonstrated using this model that additional chromatin in the "long" telomeres hybridizes actively with 3H-labelled telomere-associated Dm665 probe. The "short" telomeres show far weaker hybridization, if any, with Dm665. This means that morphological changes in the telomeres of polytene chromosomes result from different degree amplification of the telomere-associated sequences.

[A family of repeated sequences in Drosophila genome with telomeric localization: properties and polymorphism]. / Semeistvo povtoriaiushchikhsia posledovatel'nostei genoma drozofily, imeiushchee telomernuiu lokalizatsiiu: svoistva i polimorfizm.

The previously cloned Drosophila genome fragment Dm665 (2.4 kb) hybridizing with telomers on polytene chromosomes is a representative of the family of repeats, a part of which being organized in tandem clusters. The repeats are not transcribed in cell culture, are species-specific and represented in 200-250 copies per haploid genome. In natural and laboratory Drosophila lines polymorphism has been revealed with regard to homology with Dm665 in the telomeres.

[The variability of the telomeric and of a number of internal regions of Drosophila chromosomes according to the intensity of hybridization in situ with labelled probes]. / Variabel'nost' telomernykh i riada vnutrennikh raionov khromosom drozofily po intensivnosti gibridizatsii in situ s mechenymi zondami.

Cloned Drosophila DNA fragments containing telomere-specific sequences have been hybridized in situ. It is found that intensity of their hybridization with telomeres greatly varies in different nuclei of the same salivary gland. This phenomenon is also observed for internal sites where some mobile elements included in several DNA fragments under investigation are located. Within each nucleus different regions are hybridized non-uniformly as well. It is suggested that these phenomena can be explained by varying polytenization in telomeres and some internal chromosomal regions.

[Isolation of DNA fragments of the barley genome, supporting autonomous regulation of plasmids in Saccharomyces cerevisiae]. / Vydelenie fragmentov DNK genoma iachmenia, podderzhivaiushchikh avtonomnuiu replikatsiiu plazmid v drozhzhakh-sakharomitsetakh.

BamHI fragments of the barley genomic DNA were cloned in Escherichia coli cells on the vector plasmid YIp5 carrying the URA3 gene of Saccharomyces cerevisiae. Yeast cells were transformed by individual plasmid DNA preparations from each clone selected. Approximately 10% of the studied plasmids are able to replicate in yeast cells. Five barley DNA fragments which could support replication of recombinant plasmids in yeast cells belong to moderately repeated DNA and are dispersed through the chromosomes.

[Localization in the P-element of Drosophila melanogaster of sequences ensuring the autonomic replication of plasmids in yeasts]. / Lokalizatsiia v P-élemente Drosophila melanogaster posledovatel'nostei, obespechivaiushchikh avtonomnuiu replikatsiiu plazmid v drozhzhakh.

Two sequences (ARS) capable of maintaining the autonomous replication of plasmids in yeast cells were localized in the right part of Drosophila melanogaster P-element subcloned from the pi 25.1 plasmid. An ARS was found in the DNA region of genome adjacent to P-element. ARS sequences contain imperfect (10 out of 11) consensus typical of yeast ARS and have a complicated domain structure.

[Structure of the ARS element from telomeres of Drosophila melanogaster]. / Struktura ARS-élementa iz telomer Drosophila melanogaster.

We have determined the nucleotide sequence of 0.7 kb Drosophila DNA fragment specifying autonomous replication of plasmids in yeast. As shown by deletion mapping, the ARS consists of at least two domains: the core possessing the classical 11-member sequence 5'-TAAATATAAAT and the enhancer of no more than 92 nucleotide pairs located at the 3' end of the core. While comparing the sequence of the enhancer with those of 14 different ARS, we failed to detect essential homology regions. ARS elements' flanks, adjacent to the core that serve as enhancer, most probably have no regions detectable as consensus. It is suggested that they are responsible for the peculiar features of DNA secondary structure (bending, for instance) which are necessary for the interaction of proteins with ARS elements.

[Expression of cloned genes in pro- and eukaryotic cells]. / Ekspressiia klonirovannykh genov v kletkakh pro- i éukariot.

Expression of cloned genes in new environment is reviewed. Gene expression is possible under control of their own regulatory elements in the cells of related organisms. Genes may also function in cells of taxonomically remote organisms; for example, the genes of lower eukaryotes are active in bacterial cells, the Drosophila gene -- in yeast cells. The main principles of construction of pro- and eukaryotic vectore capable to provide the expression of DNA sequences in corresponding recipient cells are discussed.

[Mutation slowing down the degradation of the beta beta'-subunits of Escherichia coli RNA-polymerase]. / Mutatsiia, zamedliaiushchaia degradatsiiu beta beta'-sub"edinits RNK-polimerazy Escherichia coli.

The rpoC1 ts mutation affecting the RNA polymerase beta' subunit accelerates synthesis of RNA polymerase beta beta' subunits at 42 degrees C, while the surplus amount of subunits degrades in an hour's time. In a Ts strain with two RNA polymerase mutations, rpoC1 and rpoB251, we obtained a ts+ reversion designated opr24 which slows down degradation of surplus beta beta' subunits. The slowing down of degradation and the resulting accumulation of beta beta' subunits does not affect the kinetics of beta beta' subunit synthesis after the transfer to 42 degrees C. The effects of the opr24 are allele non-specific. The mutation also slows down degradation of beta' subunit and the amber fragment of beta subunit in the strain with subunit amber mutation rpoB22. Besides, the opr24 mutation reduces proteolysis of anomalous proteins containing canavanine. The opr24 mutation has been mapped between 17 and 21 minutes on the Escherichia coli map.

[Mutation affecting the rate of RNA-polymerase beta beta'-subunit synthesis in Escherichia coli]. / Issledovanie mutatsii, vliiaiushchei na skorost' sinteza beta beta'-sub"edinits RNK-polimerazy Escherichia coli.

As shown previously, rpoC1 temperature sensitive mutation affecting RNA polymerase beta' subunit, results in acceleration of beta beta' subunit synthesis at 42 degrees C. Also, it leads to the change in RNA polymerase sedimentation coefficient and to the reduction of RNA polymerase activity in vitro. According to the existing hypotheses, these properties of a mutant enzyme may be the cause of acceleration of synthesis of RNA polymerase in vitro. The opr1 mutation was found among Ts+ revertants of the Ts double mutant carrying rpoC1 and rif-r rpoB251 mutations. Reduction of the rate of beta beta' subunit synthesis caused by the opr1 mutation, is related to the structural changes of RNA polymerase itself. It has been shown, however, that the low activity in vitro and the altered sedimentation coefficient of the enzyme are not responsible for the accelerated synthesis of beta beta' subunits at 42 degrees C in the RpoC1 strains. Opr1 is a dominant mutation and it is located in the rpoBC region of the genetic map of Escherichia coli chromosome.

[Expression of eukaryotic genes in Escherichia coli cells]. / Proiavlenie genov éukariot v kletkakh Escherichia coli.

The paper presents a survey of literature concerned with the possibility of expression of plasmid-clones genes from eukaryotic organisms in bacteria cells. Studies on bacterial synthesis of somatostatin, human insulin, hormone of rat growth and proteins: chicken ovalbumin and mouse dihydrofolate reductase are discussed.

[New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]. / Novyi transdutsiruiushchii fag s genami beta- i beta'-sub"edinits RNK-polimerazy, proiskhodsshchii ot gibridnogo faga lambda att80: poluchenie, geneticheskii analiz i fizicheskoe kartirovanie.

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.

[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.

[Effect of several factors on synthesis of RNA-polymerase subunits by Escherichia coli K12]. / Vliianie nekotorykh faktorov na sintez sub''edinite RNK-polimerazy Escherichia coli K12.

In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits--rpoB and rpoC--the rate of synthesis of beta- and beta'-subunits is 2 times higher than in haploid cells. Missense mutation rpoC1 (tsX) in the beta'-polypeptide gene accelerates the synthesis of both beta- and beta'-subunits, particularly at a nonpermissive temperature. When rpoB-rpoC operon containing mutation rpoC1 is duplicated no dose effect of these genes is observed. In the heterozygous state mutation rpoC1 produces almost no accelerating effect on the synthesis of RNA polymerase subunits i. e. is recessive with respect to the wild allele of rpoC. In the presence of rifampicin the synthesis of RNA polymerase subunits in a sensitive wild-type strain is stimulated 6-fold, the same effect is observed with cells carrying mutation rpoC1, the latter, however, itself accelerates the synthesis of these subunits 3-fold. Thus the effects of rifampicin and the mutation are synergistic indicating that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 amber-mutant. In UV-irradiated cells, amino acid incorporation into beta- and beta'-subunits declines more rapidly than into the total protein. When either irradiated or non-irradiated cells are infected with a transducing phage lambdarifd-47 which carries rpoB gene, the synthesis of beta-proceeds at a higher rate. Irradiation of bacteria before the infection (500 erg/mm2) results in 6.5-fold acceleration of the synthesis induced by subsequent infection with lambdarifd-47 as compared to non-infected non-irradiated cells; the fraction of newly formed beta-polypeptide with respect to total protein grows 20-fold in this case. The data are considered with regard to the possible mechanisms of regulation of synthesis of RNA polymerase subunits.

[Sex factor F of Escherichia coli K12 and its participation in mobilizing bacterial chromosomes]. / Polovoi faktor F Escherichia coli K12 i ego uchatie v mobilizatsii bakterial'noi khromosomy.

The structural and functional organization of F-factor reviewed and the physical map of F DNA, supplemented with a list of genetic markers, is presented. The DNA transfer during the conjugation is considered and especial attention is given to F-gene functions involved in this process. The special sequences of F DNA, homologous to resident insertion sequences in bacterial DNA are described and its participation in plasmido-chromosomal recombinant events both dependent and independent on recA function is discussed. The mechanism for chromosome mobilization by F-Factor are reviewed. A possibility of chromosome transfer without F DNA insertion is considered. In a latter case it proposed that spontaneous single-strand breaks may serve as the origins for initiation of chromosomal transfer.

[Synthesis of RNA polymerase subunits in Escherichia coli mutants]. / Sintez cub'edinits RNK-polimerazy u mutantov Escherichia coli.

The influence of mutations in structural genes of beta- and beta'-subunits of RNA polymerase upon the synthesis of these subunits in E. coli cells have been investigated. An amber-mutation ts22 in the beta-subunit gene decreases the intracellular concentration of this subunit and the rate of its synthesis. At the same time the concentration and the rate of beta'-subunit synthesis is increased. These facts suggest the compensating activation of the synthesis of RNA polymerase subunits that takes place under the conditions of deficiency in one of the subunits. Reversions, as well as more effective suppression of ts22 amber-mutation achieved by streptomycin addition or substitution of su2 by sul result in a rise in the concentration and the rate of beta-subunit formation. This is accompanied by a drop in the concentration and the role of beta'-subunit synthesis. tsX missense motation in the beta'-subunit gene alters the properties of the enzyme increasing at the same time the concentration and the rate of synthesis of both subunits, particularly at nonpermissive temperature. This points to an inversely proportional relationship between the rate of synthesis of RNA polymerase subunits and total intracellular activity of the enzyme. Extra subunits are rapidly degraded in ts22 and tsX mutants.

[Reversion of RNA-polymerase mutations affecting F'-factor stability]. / Reversii RNK-polimeraznykh mutatsii, vliiaiushchie na stabil'nost' F'-faktorov

Ts+ reversions of amber mutations tsR on the beta-subunit of Escherichia coli RNA polymerase (79 min on the genetic map) were investigated. TsR mutants are viable due to the partial suppression of amber mutations by su2 suppressor. Three types of reversions were isolated in the course of the selection for Ts+ character, namely, intragenic reversions and two types of extragenic reversions, located at different regions of the bacterial chromosome. The mutation N5, located between 0 and 15 min. on the map, increases practically to normal the amount of RNA polymerase beta-polypeptide, which is diminished as a result of ts22 amber mutation action, and increases the plating efficiency of several T4 phage amber mutants. The mutations designated as D are located on the chromosome near the spcA locus (64 min. on the E. coli map). These mutations are characterized by pleiotropic effect. They have properties of a weak suppressor increasing the efficiency of su2 action on amber mutations of E. coli and phage T4. At the same time D mutations are capable to decrease sharply the efficiency of the crosses with F' strains. Both these characters determined by D mutations do not segregate in transduction. It is suggested that the decrease of sexduction by D mutations depends on their influence on replication of episomes in the recipient cells.