[Cloning of the RIB7 gene encoding the riboflavin synthase of the yeast Pichia guilliermondii]. / Klonirovanie gena RIB7, kodiruiushchego riboflavinsintazu drozhzhei Pichia guilliermondii.

The RIB7 gene encoding the enzyme of the final stage of riboflavin biosynthesis in Pichia guilliermondii--riboflavin synthase was cloned on the pFL38 shuttle vector as the Sau3A fragment of the chromosomal DNA of about 4 kb. The HindIII fragment of 1.4 kb was subcloned from the hybrid plasmid pFR7 obtained onto the pUC18 plasmid. The plasmid pR7 thus constructed transform Escherichia coli ribB-45 mutant cells with a blocked riboflavin synthase approximately at the same frequency as pFR7. High riboflavin synthase activity was discovered in the E. coli transformants carrying pR7 but not pFR7. Using both plasmids we also complemented rib17 mutant of P. guilliermondii.

[Cloning and inactivation of a chromosomal copy of the imidazole glycerophosphate dehydratase (HIS) gene from Hansenula polymorpha]. / Klonirovanie i inaktivatsiia khromosomnoi kopii gena imidazolglitserofosfatdegidratazy (HIS) Hansenula polymorpha.

A gene for imidazole glycerophosphate dehydratase (HIS) has been selected from the library of Hansenula polymorpha genes by complementation of Escherichia coli hisB mutations or his3 mutation of Saccharomyces cerevisiae. Inactivation of the gene in Hansenula polymorpha strain 48V, as well as Leu(-)-Leu+ conversion of phenotype and arising of antibiotic G418 resistance resulted from transformation of the strain by the linear DNA molecules of the cloned HIS-gene with the in vitro inserted LEU2 gene from Saccharomices cerevisiae and KmR gene. The Southern hybridization analysis of the Leuf+His- G418R clones representing 1-2% of transformants population together with the DNA analysis of the monospore clones from the hybrid Leu+His-G418R transformant tetrad and tester strain have revealed the locus specific nature of the clones.

[Cloning of the RIB1 gene coding for the enzyme of the first stage of flavinogenesis in the yeast Pichia guilliermondi, GTP cyclohydrolase, in Escherichia coli cells]. / Klonirovanie gena RIB1, kodiruiushchego ferment pervogo étapa flavinogeneza u drozhzhei Pichia guilliermondi--GTF-tsiklogidrolazu, v kletkakh Escherichia coli.

The RIB1 gene encoding the enzyme of the first stage of the yeast Pichia guillermondii-GTP-cyclohydrolase- was cloned on pFL38 shuttle vector as the Sau3A fragment of chromosomal DNA of about 9 kb. EcoRI fragment of 4 kb with RIB1 gene was subcloned from the pFRI hybrid plasmid obtained into the pUC18 plasmid and then shortened to give 2.9 kb via deletion in SalGI site. The plasmid constructed was designated pR1. Activity of GTP-cyclohydrolase was 80-100-fold higher in extracts of transformants than in the prototroph strain, which evidence of effective expression of the yeast gene within recombinant plasmids in the cells of this species of bacteria. The enzyme isolated from transformants has molecular mass 179 kDa, is inhibited by PAD and adenyl-nucleotides, which is characteristic of GTP-cyclohydrolase of P. guilliermondii but not of Escherichia coli.

[Riboflavin auxotrophs of Escherichia coli]. / Riboflavinovye auksotrofy Escherichia coli.

Escherichia coli riboflavin auxotrophs having a different level of riboflavin requirement were isolated. This auxotrophic mutations are located near cysB93 and trpA62 markers. The complementary effect of Bacillus subtilis riboflavin operon linked with pPR1 hybrid plasmid with rib8-1 and rib1-1 mutations was obtained.

[Genetic heteroduplex analysis of the R6K plasmid]. / Geneticheskii geterodupleksnyi analiz plazmidy R6K.

The results of genetic studies of R6K Tra1- and R6Kdelta[Sm1] mutants of R6K plasmid and those of heteroduplex analysis of DNAs have shown that DNA of this drug-resistant factor contains three loops flanked by the inverted repeats. The latter are designated as IR1, IR2 and IR3 and are of 50, 100 and 120 nucleotides in size respectively. IR1 is inserted into the loop flanked by IR2. Loops with these two repeats are located in major EcoR1 fragment, IR3 having been found in minor EcoRI fragment of the plasmid. The evidence obtained from the analysis of heteroduplex R6K/RSF2124 has shown that the loop with IR1 is corresponding to transposon Tn3. The extent of the deletion deltaSm1 indicates that IR2 may be a part of a transposon bearing the resistance to streptomycin. By comparing present data with those obtaine from the analysis of the RSF1040 factor of DNA replication initiation sites (Grosa et al., 1976), it has been suggested that the loop with IR3 represents a transposon with replicative functions (TnRep). The deletion of the mutant plasmid R6Kdelta[Sm1] (7.2 . 10(6) daltons in size) which affected one of the EcoRI sites not only confers the sensitivity to streptomycin but enhances also the efficiency of conjugational transfer and results in the loss of the R6K ability to bring about integrative suppression and to inhibit the fertility of the plasmids from IncP and IncN groups. The deletion mutant proved to have lost the property of incompatibility with the initial plasmid R6K and with itself.

[Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]. / Amplifikatsiia genov riboflavinovogo operona Bacillus subtilis v kletkakh Escherichia coli.

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.

[Eremothecium ashbyii mutants resistant to 2,6-diaminopurine]. / Mutanty Eremothecium ashbyii, ustoichivye k 2,6-diaminopurinu

3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

[Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine]. / Mutanty Eremothecium ashbyii, ustoichivye k 8-azaguaninu. Soobshchenie II. Mutanty s razlichnoi stepen'iu ustoichivosti k 8-azaguaninu

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.

[Preparation of functioning recombinant (hybrid) DNA molecules in vitro (genetic engineering experiments)]. / Poluchenie funktsioniruiushchikh rekombinantov (gibridnykh) molekul DNK in vitro (éksperimenty po gennoi inzhenerii)

In this paper we describe the preparation of hybrid plasmid consisting of ColE1 DNA and DNA of R6K plasmid. ColE1 plasmid represents a circular DNA with the molecular weight of 4,2-10(6) daltons. It determines colicine synthesis E1, has relaxed control of replication and is present in the cell in several dozens copies. Transmissive plasmid B6K represents a circular DNA molecule with the molecular weight of 28-10(6) daltons. It confers the resistance to amplicillin and streptomycin and belongs to the compatibility group X. This plasmid also has relaxed control of replication (up to 30 copies per cell). Circular superhelical DNA of ColE1 was prepared after it is amplification with cloramphenicol according to Clewell by ultracentrifugation in CsCl-EtBr density gradient. The yield of superhelical ColE1 DNA in our experiments was up to 300 mg/gram cells. Circular superhelical R6K DNA was isolated from E. coli strain J53 R6K grown to stationary phase in the presence of 15 mg/ml of streptomycin. The yield of superhelical DNA was equal to about 45 mg per 1 gram cells. EcoR1 restrictase was prepared from E. coli KM182 as described by Thomas et al. From 37 grams of cells 10 ml of enzyme solution was prepared. 1 ml of this solution was able to restrict completely 2 mg of ColE1 DNA during Hu incubation for 30 degrees min at 37 degrees C. Restrictase activity was analyzed by electrophoresis of ColE1 DNA restricts in 0,7% agarose gel as described by Tanaka. DNA ligase was prepared from E. coli B. cells infected by T4 phage am81 mutant as described by Weiss. The activity of enzyme solution was equal to 90 units/ml (according to Richardson). In standard "hybridization" experiment 4 mug of ColE1 DNA and 4 mug of R6K DNA were incubated for 1 hour at 37 degrees in 0,2 ml of the solution containing 40 mM tris-HCl, pH 7,4; 10 mM MgCl2 50 mM NaCl, 10 mM mercaptoethanol and 5 mul of EcoR1 restrictase. The reaction was terminated by heating the reaction mixture to 65 degrees C for 5 min. In accordance with the results of others CoLE1 DNA gave one restrict and R6K DNA-two restricted fragments. 100 mul of the restricted mixture was chilled to 0 degrees and the following ingredients were added 10 mul of 50 mM MgCl2; 10 mul of 100 mM dithiotreitol; 10 mul of 0,5 mM ATP, 20 mul of water and 8 mul of ligase solution (90 units/mul). The mixture was incubated at 0-0,5 degrees for 53 hours, thereafter it was used for the transformation of E. coli C600 according to Tanaka et al. Several classes of hybrid molecules due to different combinations of restricted fragments of parental species were expected. These molecules had to confer the immunity to colicine E1 and to carry the defect in the gene controlling colicine synthesis since EcoR1 restrictase splits ColE1 DNA in the region of corresponding gene. In addition, certain classes of transformants with hybrid DNA molecules had to be resistant to one or both antibiotics as determined by the R6K plasmid portion...