Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.     

Nematode Mitochondrial DNA: Anomalies and Applications

The mitochondrial genome of animal cells is currently under intense investigation by molecular, evolutionary, and population biologists. This review summarizes the available information on the molecular biology of nematode mitochondrial DNA and explains how the fundamental knowledge obtained from these basic studies may be applied to nematode systematics, evolution, and diagnostics.     

Organization and structure of Volvox beta-tubulin genes

Summary Genomic clones encoding two Volvox -tubulin genes have been isolated and shown to represent the only two -tubulin genes in the genome. Restriction fragment length polymorphism analysis was used to demonstrate that the two genes are genetically linked. One of these genes was sequenced and the mRNA start site(s) determined by primer extension. A comparison of its sequence to those of the two -tubulin genes of Chlamydomonas revealed: (1) a high degree of conservation of the coding region, with the predicted amino acid sequence differing only in the C-terminal residue; (2) extensive sequence conservation in the 5 untranslated leader region and a 16 bp (putative regulatory) sequence in the promoter region; (3) the same number and location of introns, with a short region of homology in intron 1, but little significant homology in introns 2 and 3.     

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

Summary The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by DNA-DNA hybridization of labeled probes to total DNA at various stage of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative difference in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentatively proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Secretion and export of IGF-1 in Escherichia coli strain JM101

Summary The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.