Differential Postnatal Development of Monoamine Oxidases A and B in the Blood-Brain Barrier of the Rat

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [³H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [³H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Transductional analysis of chromosome replication time

Summary Following transduction of exponentially growing cultures of Escherichia coli with phage P1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time. We show that transductants for markers located at different positions on the chromosome begin to increse at different times, in reverse order to that in which they are replicated. The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relationship to calculate the C-period of E. coli K12 growing at 30°C. We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited.     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.