Undegraded Form Research Articles

The molecular basis of codominant expression of the histocompatibility-2 genetic region.

H-2 alloantigens in an intact, undegraded form can be solubilized by the nonionic detergent NP-40 and isolated by indirect immunoprecipitation and electrophoresis in sodium dodecyl sulfate on polyacrylamide gels. With the use of an immunological precipitation method, the question about the number of cellular H-2 gene products in heterozygous cells has been partially resolved. At least four separable gene products were detected in cells heterozygous at the H-2 genetic region-one for each of the H-2D genes of the two parental haplotypes, and one for each of the H-2K genes of the two parental haplotypes. This is in contrast to the two H-2 gene products reported previously for cells homozygous at the H-2 region. The findings establish that the two parental H-2 haplotypes on homologous chromosomes in heterozygous cells are ultimately expressed as different glycoprotein molecules.

Direct activation of DNA template in lymphocyte nuclei treated with phytohemagglutinin

Nuclei from resting lymphocytes were isolated in apparently pure and undegraded form. When exposed to phytohemagglutinin for one hour the rate of DNA directed RNA synthesis in these media doubled. Furthermore, PHA treatment of resting nuclei also resulted in increased actinomycin D binding suggesting the liberation of additional segments of free chromatin. A parallel PHA-induced increase in the incorporation of 14C acetate (independent of protein synthesis) into acid insoluble nuclear products suggested a simultaneous augmentation in histone acetylation. These data demonstrate a direct stimulatory effect of PHA on the nuclei of resting lymphocytes.

The composition of the cell wall of Fusicoccum amygdali.

1. The cell wall of Fusicoccum amygdali consisted of polysaccharides (85%), protein (4-6%), lipid (5%) and phosphorus (0.1%). 2. The main carbohydrate constituent was d-glucose; smaller amounts of d-glucosamine, d-galactose, d-mannose, l-rhamnose, xylose and arabinose were also identified, and 16 common amino acids were detected. 3. Chitin, which accounted for most of the cell-wall glucosamine, was isolated in an undegraded form by an enzymic method. Chitosan was not detected, but traces of glucosamine were found in alkali-soluble and water-soluble fractions. 4. Cell walls were stained dark blue by iodine and were attacked by alpha-amylase, with liberation of glucose, maltose and maltotriose, indicating the existence of chains of alpha-(1-->4)-linked glucopyranose residues. 5. Glucose and gentiobiose were liberated from cell walls by the action of an exo-beta-(1-->3)-glucanase, giving evidence for both beta-(1-->3)- and beta-(1-->6)-glucopyranose linkages. 6. Incubation of cell walls with Helix pomatia digestive enzymes released glucose, N-acetyl-d-glucosamine and a non-diffusible fraction, containing most of the cell-wall galactose, mannose and rhamnose. Part of this fraction was released by incubating cell walls with Pronase; acid hydrolysis yielded galactose 6-phosphate and small amounts of mannose 6-phosphate and glucose 6-phosphate as well as other materials. Extracellular polysaccharides of a similar nature were isolated and may be formed by the action of lytic enzymes on the cell wall. 7. About 30% of the cell wall was resistant to the action of the H. pomatia digestive enzymes; the resistant fraction was shown to be a predominantly alpha-(1-->3)-glucan. 8. Fractionation of the cell-wall complex with 1m-sodium hydroxide gave three principal glucan fractions: fraction BB had [alpha](D) +236 degrees (in 1m-sodium hydroxide) and showed two components on sedimentation analysis; fraction AA(2) had [alpha](D) -71 degrees (in 1m-sodium hydroxide) and contained predominantly beta-linkages; fraction AA(1) had [alpha](D) +40 degrees (in 1m-sodium hydroxide) and may contain both alpha- and beta-linkages.

Effects of elevated temperatures on mengovirus ribonucleic acid synthesis and virus production in Novikoff rat hepatoma cells.

The production of mengovirus in Novikoff rat hepatoma cells is progressively reduced with an increase in incubation temperature of the cells from 34 to 40 C, in spite of the fact that about the same amounts of single-stranded and double-stranded viral ribonucleic acid (RNA) are synthesized at 34, 37, and 40 C; the rate of overall protein synthesis is as high at 40 C as at 37 C. At 40 C, progeny viral RNA accumulates in an undegraded form without being incorporated into virus particles. The results suggest that virus maturation is preferentially inhibited at supraoptimal temperatures. At 42 C, on the other hand, no viral RNA is produced and no viral RNA polymerase activity is detectable in cell lysates. Failure of infected cells to form viral RNA polymerase at 42 C is probably due to an impairment of protein synthesis since most of the polyribosomes are rapidly lost during incubation at 42 C and the rate of amino acid incorporation into protein is 70% lower at 42 C than at 37 C. When infected cells are shifted from 37 to 42 C during the period of active viral RNA synthesis, viral RNA polymerase activity is rapidly lost from the cells, and viral RNA synthesis ceases within 45 min. In contrast, the RNA polymerase is as active in vitro at 42 C as at 37 C, and the activity is relatively stable at 42 C.

Degradation of cell constituents by starved Streptococcus lactis in relation to survival.

SUMMARY Streptococcus lactis organisms were grown in lactose-limited batch culture and transferred, after washing, to phosphate buffer at the growth temperature. Soluble protein was released from viable organisms into the suspending buffer and the intracellular free amino acid pool declined steadily with the components appearing in the suspending buffer; a net increase in the total amount of free amino acid indicated some protein hydrolysis. RNA was hydrolysed, resulting in the release of u.v.-absorbing bases and ribose from the organisms. Conditions which promoted rapid RNA breakdown also produced rapid death rates and long cell division lags in surviving organisms. After 28 hr starvation in buffer containing Mg2+, the bacterial dry wt decreased by 26 %; loss of RNA, protein and free amino acids accounted for 10·3 %, 7·3 % and 2·7 % of the total bacterial mass loss. The products of polymer hydrolysis appeared to be released in an undegraded form into the external buffer and there was no appreciable formation of lactate, ammonia or volatile fatty acids, possibly indicating the absence of any important endogenous energy sources. There was no appreciable degradation of DNA or carbohydrate but phospholipid was broken down on prolonged starvation. No polyglucose or poly-β-hydroxybutyrate was detected in the organisms.

Studies on the formation of tobacco mosaic virus ribonucleic acid. 8. Fate of parental viral RNA in the host cell.

Fate of parental viral RNA in the host cell was determined with the use of P(32)-labeled tobacco mosaic virus (TMV)-RNA as inoculum. There was a wide variation in the retention of TMV-RNA that was rubbed on the tobacco leaf surface. On the average, about 0.73 mug of viral RNA was retained by tobacco leaf. About 63 per cent of the retained RNA was degraded in the leaf cells. On the average, about 0.27 mug of TMV-RNA was conserved in a tobacco leaf. The conserved parental TMV-RNA was remarkably stable and was located in the nuclear fraction of leaf homogenate. It could be isolated in an undegraded form and was RNase-sensitive, presumably single-stranded. About 2 per cent of it was associated with progeny TMV.

Translation of poliovirus RNA by an E. coli cell-free system.

Poliovirus RNA is an ideal mammalian mRNA for in vitro studies of protein biosynthesis because it can be prepared in fairly large quantities in an undegraded form from virions. It is known to direct the synthesis of several poliovirus proteins in infected HeLa cells. The tryptic peptides of these proteins can be identified (Jacobson, Asso, and Baltimore, in prep.; Baltimore, Jacobson, Asso, and Huang, this volume) and compared with those made in vitro. In vivo, it apparently codes for only a single polypeptide which is subsequently cleaved to form the functional proteins (Jacobson and Baltimore, 1968b; Baltimore et al., this vol.).

Identification and estimation of undegraded thyroglobulin in human serum.

A radioimmunoassay for the detection of thyroglobulin in human serum having a sensitivity of 10 ng/ml is described showing that conventional techniques can be applied to the assay of large globular proteins. In 60% of normal sera the assay was positive. The serum of athyreotic cretins gave negative results. The active material exhibited the same behavior as native thyroglobulin when assayed at multiple dilutions and when subjected to ultracentrifugation and electrophoresis; furthermore, it could be absorbed out with insoluble preparations of rabbit or human antithyroglobulin antibodies. It was concluded that the assay was specific for thyroglobulin and that this protein is commonly present in an undegraded form in normal human serum. The implications of these findings are discussed in relation to theories of auto-immunity. (Endocrinology 81: 421, 1967)

The isolation, composition and physicochemical properties of deoxyribonucleic acid from Bordetella pertussis

The percentage by weight of deoxyribonucleic acid in Bordetella pertussis is large. A method is described for the isolation of DNA from this bacterium in a relatively pure and undegraded form. DNA was extracted from several strains and had a base composition of 67.6% GC 2 2 % GC = number of moles of (guanine + cytosine) per 100 moles of (adenine + thymine + guanine + cytosine). ; in one case, however, the composition was 57.5% GC. The two types of DNA were studied further: they contained no large amounts of bases other than the usual four, their light-scattering and sedimentation properties were very similar, and their thermal denaturation properties were consistent with the difference in base composition.

INTRACELLULAR FATE OF MENGO VIRUS RIBONUCLEIC ACID.

Homma, M. (The Wistar Institute, Philadelphia, Pa.), and A. F. Graham. Intracellular fate of Mengo virus ribonucleic acid. J. Bacteriol. 89:64-73. 1965.-P(32)-labeled, purified preparations of Mengo virus adsorbed rapidly and irreversibly to L cells maintained in suspension cultures. At intervals after adsorption of labeled virus, the total ribonucleic acid (RNA) of infected cells was extracted by a phenol technique. Infectivity titrations on this RNA showed that it retained its full biological activity during the early part of the eclipse period. Sucrose gradient sedimentation analyses showed also that this RNA lost none of its structural integrity throughout the eclipse period. No evidence was found for a double-stranded structure involving parental RNA. When cells infected with P(32)-labeled virus were broken open during the first 7 hr after infection, no more than 20% of the parental RNA could be digested with ribonuclease. Electron microscopy indicated that only one particle in five of the purified viral populations was infectious. It is suggested that only one of five adsorbed virus particles was uncoated and that its RNA remained in the cell in an undegraded form during the eclipse period. The other adsorbed particles were not uncoated and took no part in the process of infection. The maximal transfer of parental RNA to progeny virus was 4.5%.