Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice.

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a nonmalignant tissue. The degree of proviral amplification is small (ca. one to five additional copies) and appears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggest a clonal origin for the tumor mass. In addition, the restriction patterns observed after digestion with the enzymes BglII and SacI indicate that only one of the proviruses endogenous to GR mice is amplified. The amplified provirus found in GR mammary tumors is identical to the provirus that is missing in GR-Mtv-2- mice, a congenic line exhibiting a low mammary tumor incidence.     

Prolactin receptors in mammary tumors of GR mice.

Prolactin receptors have been identified in estrone-progesterone induced mammary tumors from GR mice. 125I-labeled ovine prolactin binding to tumor homogenates reached a steady state in 12 hours at 22 degrees and was specific for prolactin. Prolactin receptors were highest (16 fmoles/mg protein) in primary, hormone-dependent tumors and declined progressively in transplanted hormone-dependent and transplanted hormone-responsive tumors. In autonomous tumors, binding was approximately 5% of that found in primary tumors. Scatchard analysis of binding to selected tumors indicated that the observed decrease in bound hormone was due to a loss in the number of receptor sites; binding affinity was unaltered (kd approximately 1 X 10(-10) M). Since receptors for estrogen and progesterone as well as those for prolactin decline in a concerted manner with the transition to autonomy, autonomous growth may result from a loss of receptors or an increase in the relative proportion of autonomous cells present in the tumor.     

Increased isoleucine acceptance by sulfur-deficient transfer RNA from Escherichia coli.

Sulfur-deficient tRNA, isolated from Escherichia coli HfrC, rel-, met-, cys-, lambda, after cysteine starvation, was found to have an increased acceptance of isoleucine in proportion to the deficiency of 4-thiouridine. Isoleucine acceptance was not altered in the presence of other amino acids of CTP, and the higher acceptance was observed over a wide range of magnesium, isoleucine, tRNA and enzyme concentrations. The Vmax value for sulfur-deficient tRNA was more than three times greater than observed for normal tRNA. Methylated albumin kieselguhr (MAK) chromatography revealed three isoacceptor peak for normal tRNA, while sulfur-deficient tRNA was missing tRNAile, and exhibited a larger, shifted peaks for tRNA normal tRNA, while sulfur-deficient tRNA was missing tRNAille 2, and exhibited a large shifted peak for tRNAile 3 . Treatment with crude RNA sulfurtransferase both lowered the isoleucine acceptance for sulfur-deficient tRNA to that seen for normal tRNA, and restored the missing isoacceptor on MAK. The possibility that thionucleotides may play a role in the aminoacylation of tRNAile in E. coli is discussed.     

Relationship between changes in the translational apparatus and actinomycin production in Streptomyces antibioticus.

As previously reported (G. H. Jones, 1975), transfer ribonucleic acids (tRNA's) and ribosomes from actinomycin-producing cultures of Streptomyces antibioticus show a decreased ability to function in aminoacylation and translation as compared with the corresponding components from younger cells. Further, specific changes in the isoacceptor patterns are revealed when tRNA's from actinomycin-producing cells are compared with those of younger cells by reverse- phase column chromatography. A specific glycyl-tRNA species is eliminated from the reverse-phase profile of tRNA's from actinomycin-producing S. antibioticus cells as compared with younger cells. Changes in isoacceptor patterns were also observed for the amino acids methionine, valine, phenylalanine, and leucine. Actinomycin synthesis was inhibited by growing S. antibioticus cells in the presence of alpha-methyl-DL-tryptophan. Inhibition of actinomycin synthesis reversed the changes in tRNA observed in normally grown control cultures, although it had no demonstrable effect on the growth of the cells. Thus, tRNA from 48-h-old, alpha-methyl-tryptophan-grown cells had amino acid acceptor activity that was equal to or greater than that of tRNA from 12-h-old, normally grown cells. Similarly, the reverse-phase chromatographic pattern for glycyl-tRNA's from 48-h-old, alpha-methyl-tryptophan-grown cells was identical to that of the glycyl-tRNA's from 12-h-old, normally grown cells. In contrast, the ability of ribosomes from 48-h-old, alpha-methyl-tryptophan-grown cells to function in polypeptide synthesis in vitro was essentially identical to that of 48-h-old, normally grown cells. Ribosomes from 12-h-old, normally grown cells were severalfold more active in in vitro polypeptide synthesis.     

Methylation and amplification of mouse mammary tumor virus DNA in normal, premalignant, and malignant cells of GR/A mice.

The methylation and amplification of mouse mammary tumor virus (MuMTV) proviral DNA was investigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylation pattern was examined with the restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heart was highly methylated. Proviral DNA was somewhat undermethylated in mammary gland cells from virgin and lactating mice and extensively undermethylated in cells from premalignant outgrowths, pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme SacI was used to detect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammary gland cells from virgin mice. Some mammary gland cells from lactating mice appeared to contain additional copies of the endogenous, highly oncogenic GT-MTV-2 provirus. Premalignant outgrowth, pregnancy-dependent tumor, and pregnancy-independent tumor cells contained an average of two to three additional copies per cell of the GT-MTV-2 provirus. Thus, neoplasia in GR/A mice was directly associated with quantized increases in MuMTV proviral DNA undermethylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme analysis suggested that premalignant outgrowths and pregnancy-dependent tumors both consisted largely of heterogenous cell populations, although some evidence of clonal dominance was detected.     

2'-Versus 3'-OH specificity in tRNA aminoacylation. Further support for the "secondary cognition" proposal.

Purified Escherichia coli tRNAAla and tRNALys were each converted to modified species terminating in 2'- and 3'-deoxyadenosine. The modified species were tested as substrates for activation by their cognate aminoacyl-tRNA synthetases and for misacylation with phenylalanine by yeast phenylalanyl-tRNA synthetase. E. coli alanyl- and lysyl-tRNA synthetases normally aminoacylate their cognate tRNA's exclusively on the 3'-OH group, while yeast phenylalanyl-tRNA synthetase utilizes only the 2' position on its own tRNA. Therefore, the finding that the phenylalanyl-tRNA synthetase activated only those modified tRNAAla and tRNALys species terminating in 3'-deoxyadenosine indicated that the position of aminoacylation in this case was specified entirely by the enzyme, an observation relevant to the more general problem of the reason(s) for using a particular site for aminoacylation and maintaining positional specificity during evolution. Initial velocity studies were carried out using E. coli tRNAAla and both alanyl- and phenylalanyl-tRNA synthetases. As noted in other cases, activation of the modified and unmodified tRNA's had essentially the same associated Km values, but in each case the Vmax determined for the modified tRNA was smaller.     

Study of the transfer RNAs coded by T2, T4, and T6 bacteriophages.

T2, T4, and T6 bacteriophage tRNAs coding for arginine, leucine, proline, isoleucine, and glycine were isolated under conditions of short term and long term infection of Escherichia coli B cells. The corresponding phage tRNA species were examined for sequence homology by RNA-DNA hybridization analysis and by their relative behavior on reversed phase chromatography. The results indicate that all three T-even phages code for similar tRNA species; however, some tRNA species are homologous, others are not, and not all of the same tRNA species are coded by each bacteriophage. Reversed phase chromatography showed the presence of isoacceptor tRNAs for each phage aminoacyl-tRNA species. Pulse-chase experiments for [32P]tRNAGly suggest that the multiple isoacceptor species observed derive from the intracellular modification of a single tRNAGly gene product.     

Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis.

The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

Histone phosphorylation in explants of mouse mammary glands and tumors

Rates of histone phosphorylation were measured in explants of mammary glands from mouse strains with high and low tumor incidence. Explants of hormone dependent and independent mouse mammary tumors were also investigated. All mouse strains studied showed predominant phosphorylation of H2A histone at serine and threonine residues. No differences in rates of H2A phosphorylation in glands were found between strains having different mammary tumor susceptibility. Hormone-dependent GR mouse mammary tumors also showed high H2A phosphorylation, but in some tumors also H1 and H3 were phosphorylated. Hormone-dependent GR tumors had 2–5 times higher histone phosphorylation at serine and threonine than hormone-independent tumors.     

Prolactin and progesterone receptors in pregnancy-dependent mammary tumors in GR/A mice

In this study, cellular prolactin receptors and cytosolic progesterone receptors were examined and compared in pregnancy-dependent mammary tumors (PDMT) and in normal mammary glands of pregnant GR/A mice. PDMT and normal mammary glands were examined in the same animal, thus assuring an identical hormonal environment. The PDMT cells had a larger capacity to bind prolactin or the synthetic progesterone, R5020, than did the normal mammary gland. While the dissociation constant (Kd) value for prolactin binding to normal mammary epithelial cells was similar to that of PDMT cells, PDMT cells had 2.2 times more prolactin receptors than the normal cells. Progesterone binding activity was detected only in PDMT, but not in the normal mammary cells. The receptor concentration and the Kd value for progesterone binding of PDMT were 606 fmol/mg protein and 3.53 nM, respectively. It appears, therefore, that normal regulation of these receptors may be altered within the PDMT cells. The increased growth responsiveness of PDMT to the hormones of pregnancy, especially prolactin, progesterone, and placental lactogen, may be a function of a sharp increase in the level of cellular receptors for these mammotropic hormones.     

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.     

Lysine tRNAs from Bacillus subtilis 168: function of the isoacceptors in a rabbit reticulocyte cell-free protein-synthesizing system.

The two principal tRNA Lys isoaccepting species of Bacillus subtilis were compared in their functional activity in translating rabbit globin. Although neither species demonstrates any preference in reading either of the lysine codons, there is an overall preference for tRNa Lys 3 in lysine incorporation. The ratios of lysine incorporated by the two species into the different lysine-containing sites in the globin subunits vary over a more than two-fold range. As described in the accompanying paper, tRNA Lys 1 is a hypomodified form of tRNA Lys 3. Consistent with studies on other rRNA species, the fully modified isoacceptor functions preferentially. In contrast to these results, however, the fully modified isoacceptor (tRNA Lys 3) is found predominantly in rapidly dividing cells while the hypomodified isoacceptor (tRNA Lys 1) predominates in the stationary cells and spores of B.l subtilis.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

Nafoxidine administered to newborn female GR mice arrests the development of their mammary glands

Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 beta estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands.     

Mammary tumor induction loci in GR and DBAf mice contain one provirus of the mouse mammary tumor virus

The mammary tumor induction genes Mtv-1 in mouse strain DBAf and Mtv-2 in strain GR control the complete expression of the endogenous mouse mammary tumor virus (MMTV). We have used a combination of genetic, biochemical and molecular biological methods to identify and correlate specific copies of the endogenous MMTV proviral genes with the biological properties of the tumor induction genes Mtv-1 and Mtv-2. These Mtv induction genes contain specific MMTV proviral information, as was concluded from restriction enzyme analysis and molecular hybridization of DNAs of congenic mouse strains and of progenitors of backcross populations. The congenic strains differed from the parental strains GR and 020 only in the Mtv-2 gene, one lacking the Mtv-2 gene (GR/Mtv-2-) and one having obtained this gene (020/Mtv-2+). The gain or loss coincided with two Eco RI cellular DNA fragments containing MMTV DNA information. Since Eco RI cuts the exogenous proviral variant of MMTV DNA once, we assume that these two cellular DNA fragments contain one MMTV provirus. The same cellular DNA fragments containing MMTV DNA information segregated together with MMTV expression in the offspring population of the backcross. In a similar backcross analysis of the induction gene Mtv-1 it was also demonstrated that the Mtv-1 gene comprises one MMTV provirus. These data indicate that Mtv induction genes contain specific but different MMTV proviral genes and that nly a limited number of the MMTV proviruses present in the cellular DNA is associated with the control of proviral expression.