Search PubMed⌕ Search

Biomedical subjects

H Klett

Publications and source records attributed to H Klett.

7 recordsLinked to original sources
PubMed1978

Measurement of polyadenylic acid by hybridization with polyuridylic acid: a source of error due to the lability of tritiated polyuridylic acid in trichloroacetic acid.

During brief exposure to trichloroacetic acid at 0 degree C, significant amounts of tritiated polyuridylic acid are converted to acid-soluble products. The loss of polymeric radioactivity is dependent on both acid concentration and the time of exposure to acid. When trichloroacetic acid precipitation is used to recover the tritiated polyuridylic acid present in a hybrid with polyadenylic acid, significant underestimates of the hybridized radioactivity can occur because of the lability of tritiated polyuridylic acid. Conditions are given which minimize the lability and permit quantitative recovery of tritiated polyuridylic acid by trichloroacetic acid precipitation.

Chemical Precipitation↗
PubMed1978

Decrease in functional albumin mRNA during estrogen-induced vitellogenin biosynthesis in avian liver.

Translation of rooster liver RNA in a wheat germ extract is shown to yield albumin as one of the cell-free products. Quantitation of albumin mRNA by the translation assay indicates that functional albumin mRNA represents 10% of total liver mRNA activity in control roosters. After estrogen administration, this level decreases in a continuous fashion until functional albumin mRNA represents 5% of total mRNA activity at 12 days. This decrease in functional albumin mRNA was correlated with several parameters of vitellogenin induction. Functional vitellogenin mRNA increases to a maximum at 4 days after hormone treatment and returns to control levels by 12 days. A similar pattern is seen for the hormone-stimulated increase in total mRNA activity. The decrease in functional albumin mRNA, therefore, persists after the vitellogenic response of the liver has been completed. These results suggest that the decrease in hepatic albumin synthesis after hormone treatment is due to an estrogen-mediated decrease in the content of albumin mRNA.

Albumins↗
PubMed1974

Shythesis of reovirus oligo adenylic acid in vivo and in vitro.

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 mug of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The (3)H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of approximately 45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins.

Adenosine Monophosphate↗
PubMed1971

Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro.

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.

Animals↗
PubMed1970

Properties of RNA transcriptase in reovirus subviral particles.

Subviral particles containing reovirus RNA transcriptase have been isolated from extracts of virus-infected mouse fibroblast cells. The purified particles which lacked the outer protein capsomeres of the mature virion had a buoyant density of 1.43-1.44 g/ml in CsCl and contained all of the double-stranded RNA genome of the intact virus. The particles were free of nuclease activity. RNA synthesis required all four ribonucleoside triphosphates and was dependent on magnesium or manganese; optimal activity required potassium or ammonium ions. In the presence of a ribonucleoside triphosphate regenerating system, reaction rates were linear for 20 hr. RNA yields of 40-fold in excess of input template could be obtained. Completed RNA chains were released from the subviral particles. In the course of RNA synthesis, the double-stranded RNA template was fully conserved. The RNA products formed in vitro displayed profiles in sucrose gradients similar to those found for in vitro reovirus mRNA. The RNA products were single-stranded and did not self-anneal. Over 90 percent of the transcriptase products could be annealed with template double-stranded RNA. The annealed products migrated in acrylamide gels as double-stranded RNA, indicating efficient in vitro transcription.

Centrifugation, Density Gradient↗