Some peculiarities of gene expression in inbred and hybrid rats under normal conditions and after partial hepatectomy

The peculiarities of gene expression in line and hybrid rats in the normalcy and after partial hepatectomy were studied. It was shown that the relationship between the RNA synthesis rate in chromatin and RNA transport in the cytoplasm as well as the correlation between the RNA transport rate and the activity of the protein-synthesizing mechanism vary in animals with different genotypes. After partial hepatectomy the hybrids have their S-period of the cell cycle earlier than the line animals. The hybrids do not differ from the parental forms in the rate of RNA chromatin synthesis; however, the newly synthesized RNA of the hybrids in transported into the cytoplasm 5.7 and 5 times faster than that of the lines Vistar and August. The rapidly transported RNA of hybrids is likely to have a more prolonged life as compared to the line animals. After partial hepatectomy an activation of the whole gene expression system occurs. The differences in the gene expression system of hepatocytes of inbred and hybrid animals in the regenerating liver become less pronounced.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Variations in rat liver chromatin composition during growth and the heterotic response

The manifestation of hybrid vigor with regard to the inheritance of body weight was established as a postweaning phenomenon in rats. Liver weight gain generally corresponded to body weight gain and also reflected hybrid vigor. The protein/DNA ratios of the chromatin from livers of inbred and heterotic hybrid male rats were found to increase with increasing age in growing rats. Hybrid chromatin had higher protein/DNA ratios throughout the age range studies. The difference between hybrids and inbreds were greatest in the early postweaning stages of growth and maturation. The RNA/DNA ratios of chromatin from livers of inbred and hybrid male rats were also found to exhibit trends within certain developmental periods.This investigation was supported by matching Federal and West Virginia State funds from the Hatch 190 grant and is published with the approval of the Director of the Agricultural Station as Scientific Paper No. 1293.     

Nuclease accessibility of chromatin from a heterotic hybrid and from parental inbreds

The DNAase II, Mg²⁺ procedure was used to fractionate the seedling chromatin of a heterotic maize hybrid and its parental inbreds FRM017 and FRN28. The hybrid and the more vigorous maize inbred FRN28 have 14 and 11 percent soluble chromatin (euchromatin) respectively, while the less vigorous FRM017 contains 30 percent. The unfractionated chromatin of the hybrid contains less protein than either inbred. The RNA contents of the unfractionated chromatin of the hybrid and of FRN28 are similar and are one-half that of less vigorous FRM017. Hybrid euchromatin contains relatively more protein and RNA than DNA as well as higher proportions of protein and RNA than heterochromatin, unfractionated chromatin, or inbred euchromatin; this suggests a more “efficient” type of activity, as reflected by the low amount of euchromatin and a high proportion of RNA and chromosomal proteins.     

RNA Stimulated by Indole Acetic Acid

THE stimulation of RNA synthesis in plant cells by auxins^1,2 may be due, in part, to gene derepression, since chromatin isolated from hormone-treated cells is a better template. Mathysse and Phillips³ demonstrated that an acceptor protein for auxin can interact with the chromatin to derepress the genome. In their system the hormone and protein do not affect the rate of RNA synthesis if pure DNA. is used as a template. However, the actual mechanism of auxin on RNA synthesis by isolated RNA polymerase system has yet to be elucidated.     

Alterations in myocardial RNA synthesis and RNA polymerase activity during normal growth

The effect of normal growth (hypertrophy) on myocardial nuclear activity was investigated using male Wistar rats at 21, 50, and 100 days of age. Cardiac mass increased sevenfold during this age range. The concentration of RNA (mg X g-1) was the highest at 21 days and decreased 48% by 50 days of age and 68% after 100 days of development. RNA synthesis, corrected for alterations in the specific activity of the cytoplasmic nucleotide pool, was the highest at 21 days of age. After 50 days of growth, uridine incorporation was decreased fivefold. With continual growth (100 days), RNA synthesis was still reduced compared with the 21-day animals. RNA polymerase activity in myocyte nuclei showed little change in activity from 21 to 100 days of age. However, in the nonmyocyte fraction, RNA polymerase decreased threefold after 50 days of development. Collectively, these data suggest that the large decrease in myocardial RNA synthesis cannot be accounted for by a change in nuclear RNA polymerase activity and that an alteration in chromatin template capacity may be involved during this form of cardiac growth.     

Mode of inhibition of DNA synthesis induced by adenosine diphosphoribosylation of nuclear protein

DNA obtained after complete removal of the proteins from NAD-treated rat liver chromatin possessed template capacity equivalent to that obtained from untreated chromatin. The stepwise extraction of proteins from chromatin affected a gradual removal of labeled ADPR and a parallel decrease of the inhibition. The results of the present study suggest that the inhibition of the template capacity for DNA synthesis following preincubation of chromatin with NAD is dependent upon ADP-ribosylation of the associated proteins. The template capacity of chromatin and nucleoprotein complex (Weiss preparation) for DNA synthesis was inhibited, whereas the capacity for RNA synthesis was apparently not affected.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template-free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template-translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template-containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.