Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.     

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene

Summary A method for transposon mutagenesis in Azospirillum lipoferum 29708 is reported with transposon Tn5. The suicide plasmid pSUP2021 was used to deliver Tn5 in A. lipoferum using Escherichia coli SM10 as the donor. Neomycin-resistant transconjugants were detected at a frequency of 6x10^-6 per recipient. Different types of mutants were isolated, e.g. auxotrophic, coloured, IAA-negative, and IAA-overproducers. Among the auxotrophic mutants, cysteine and methionine requirers prevailed. Random Tn5-insertion with only one copy per mutant was demonstrated by Southern blotting and hybridization. Tn5-induced mutants are relatively stable, with reversion rates of 2–20×10^-8. A gene which is a part of the carotenoid pathway is closely linked to the histidine genes. The existence of two pathways for IAA production in A. lipoferum is discussed.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Sequence instability in the long terminal repeats of avian spleen necrosis virus and reticuloendotheliosis virus

Summary Sequence divergence between the 3 long terminal repeats (LTR) of avian reticuloendotheliosis virus (REV), deletion variant proviral clone 2-20-4, and spleen necrosis virus (SNV)—proviral clones 14-44, 60, and 70—was found to involve two classes of base substitutions: low-frequency interspersed and high-frequency clustered substitutions. Clones 2-20-4 and 14-44 have diverged 4.4% owing to low-frequency substitutions. In contrast, two high-frequency substitution segments have diverged by 30% and 29%, respectively. Clustered substitutions appear to be located either within or next to tandem repeats, suggesting their introduction concomitant with sequence deletions and duplications commonly associated with such repeats. A new 19-bp tandem repeat is found in clone 2-20-4. Its sequence could have evolved from the 26-bp repeats found in the SNV clones.     

Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.