Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

The ‘A rule’ of mutagen specificity: A consequence of DNA polymerase bypass of non-instructional lesions?

The replicative bypass of lesions in DNA and the induction of mutations by agents which react with DNA to produce damaged bases can be understood on the basis of a simple kinetic model. Bypass can be analyzed by separately considering three processes: a) addition of a base opposite a lesion, b) a proofreading excision process, and c) a rate limiting elongation step. Adenine nucleotides are preferentially added opposite many lesions making it possible to predict mutational specificity. Replicative bypass (translesion synthesis) is dependent on modulation of proofreading exonuclease activity but loss of exonuclease activity alone is not sufficient to ensure bypass. Frameshift mutation is the result of the failure of translesion synthesis accompanied by rearrangement of the template, particularly at repetitive sites.     

Effects of prolactin on progestin secretion by human granulosa cells in culture

Concentrations of human prolactin (hPrl) greater than or equal to 600 ng/ml produced inhibition of progestin production in cultures of granulosa cells pooled from follicles of women stimulated with clomiphene citrate-human chorionic gonadotropin (hCG). However, cells collected from follicles of human menopausal gonadotropin (HMG)-hCG-treated patients did not demonstrate a significant reduction in progestin secretion in response to hPrl. We conclude that high concentrations of hPrl can result in inhibition of steroidogenesis, but the expression of the inhibitory effects of Prl depends upon the hormonal treatments used to stimulate follicular growth.     

Biosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa

We have examined the biosynthesis of rat apolipoprotein C-III in the small intestine and liver. The primary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Comparison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-III showed that apo-C-III was initially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu-Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post-translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-II. The mRNA encoding apolipoprotein C-III comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyceride meal resulted in a 2-fold increase in intestinal preapo-C-III mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute response of the apo-A-I and C-III genes to triacylglycerol absorption differs.     

Steps in the stabilization of newly packaged DNA during phage P22 morphogenesis

The protein products of three adjacent P22 genes, 4, 10 and 26, are required for the stabilization of DNA newly packaged into P22 phage capsids. We have isolated unstable DNA containing capsids from cells infected with mutants defective in these genes. All three classes could be converted into mature phage in vitro, confirming that they represent intermediates in particle maturation. The first of the three proteins to add to the newly filled capsids is gp4, followed by gp10 and gp26. The active form of gp4 sediments at 3 S, while the active forms of both gp10 and gp26 sediment at 5 S. These soluble subunits appear to polymerize onto the newly filled capsids to form the neck of the mature phage, the channel for DNA injection. Since gp4 is the first protein to act after DNA packaging, the unstable DNA containing capsids from 4- -infected cells must represent the direct product of the packaging of DNA into procapsids. The major fraction of these capsids lost activity with a half-life of 1.1 minutes at 23 degrees C, though they were much more stable at 0 degree C. Electron microscopic observations indicated that the loss of activity was due to the DNA exiting from the incomplete capsids. The marginal stability of the condensed DNA molecules within capsids is consistent with models of ATP-driven condensation and spontaneous DNA ejection. The basis of the stability of these highly condensed molecules remains to be determined.     

Molecular heterogeneity of creatine kinase isoenzymes

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.     

Characterization of an Infectivity Assay for the Ribonucleic Acid of Bacteriophage MS2

An infectivity assay for MS2 ribonucleic acid (RNA), which uses bacterial spheroplasts of an F(-) strain of Escherichia coli, has an efficiency of 10(-5) infected spheroplasts per RNA molecule. The characteristics of this assay and the influence of several parameters are presented. Important variables include the duration of exposure of the cells to lysozyme-ethylenediaminetetraacetic acid, the duration of exposure of the spheroplast stock to the RNA solutions before dilution, the concentration of RNA, and the presence of competing RNA. The growth kinetics of the virus in the infected spheroplasts and the extent of lysis have also been studied.