Search PubMed⌕ Search

Biomedical subjects

R Reddy

Publications and source records attributed to R Reddy.

At least 199 records · Page 11Linked to original sources
PubMed1990

Capping of U6 small nuclear RNA in vitro can be uncoupled from transcription.

U6 small nuclear RNA (snRNA) is a required component in the splicing of eukaryotic pre-mRNAs. Mammalian U6 snRNA was synthesized in vitro by T7 RNA polymerase and purified on polyacrylamide gels. This U6 RNA, with pppG on its 5' end, was accurately capped to CH3-O-pppG, when incubated with HeLa cell extract and this capping was dependent on the capping signal present within the U6 snRNA. When gamma-32P-labeled U6 RNA was used as a substrate, the U6 cap formed in vitro retained this labeled gamma-phosphate, indicating that the cap formation involves the methylation of the gamma-phosphate incorporated during transcription. U6 snRNAs with ppG or pG as their 5' ends, were not capped in this in vitro capping system. Capping of U6 snRNA in vitro requires at least two components, a heat-labile component and S-adenosylmethionine as a methyl group donor. The data presented here show that capping of U6 snRNA can be uncoupled from transcription and that the mechanism of U6 snRNA cap formation differs markedly from the capping mechanism of mRNAs and other U snRNAs where capping is cotranscriptional. While many methyltransferases have been characterized earlier, this is the first report of a methyltransferase that is specific to phosphate residues. This in vitro capping system will be useful for purification and studies on the U6 snRNA sequence-dependent methyltransferase activity.

Animals↗
PubMed1990

Rehabilitation of burn patients with concomitant limb amputation: case reports.

Burn patients with associated limb amputations present demanding rehabilitation problems, many of which might be expected to lead to chronic difficulties. Therapeutic goals following limb amputation include oedema reduction, prevention of contracture (through positioning and range of motion), stump shaping, both pre- and post-prosthetic fitting strengthening exercises of the limb and trunk, and gait training. Some patients present problems that are associated with both the burn injury and the limb amputation that cause concern among the physical therapy staff. Some of these situations include intolerance of the stump to pressure or manipulation due to remaining open wounds or fragility of newly skin grafted areas on the residual limb or delayed gait or functional training due to wounds on other body surface areas. Delays in stump preparation or other treatment aims due to continued surgical procedures can be worrisome. A review of these patients indicates the possible difficulties that rehabilitation personnel may face when treating burn victims who required amputation. Effective rehabilitation of these patients can be achieved despite the noted concerns.

Adult↗
PubMed1990

Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA.

The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R. Singh and R. Reddy, Proc. Natl. Acad. Sci. USA 86:8280-8283, 1989). Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA. The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop. Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant. Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript. Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote. The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Base Sequence↗
PubMed1990

Developmental changes of tropoelastin synthesis by rat pulmonary fibroblasts and effects of dexamethasone.

Lung elastin is an important extracellular structural protein and it has been postulated that it plays a regulatory role in alveolar formation. To study the developmental regulation of elastin gene expression, we examined the tropoelastin (TE) production in primary culture of rat pulmonary fibroblasts (RPF). We found that developmental changes in elastin production as assessed by TE synthesis and 3.6-kb TE mRNA levels were similar for RPF and whole tissue except those results from late gestation animals, with peak elastin expression occurring 7 d postnatally with a decline out to 21 d. At late gestation (20 d), TE mRNA was barely detectable in RPF but clearly detectable TE mRNA in the whole tissue, indicating that there are elastogenic cells other than RPF in the tissue at this age. When TE-producing cells were treated with dexamethasone, there was a dose-dependent stimulation of TE synthesis with the maximum response at 10(-9) to 10(-7) M. Interestingly, dexamethasone had no stimulatory effect on cells from late gestation animals. The developmental window of elastin synthesis in this RPF model between late gestation and 21 d postnatal seems to correlate with the reported period of secondary alveolar formation, and thus we speculate that RPF elastogenic activity reflects that of the alveolar wall.

Animals↗
PubMed1989

Secondary structure of 7SK and 7-2 small RNAs. Possible origin of some 7SK pseudogenes from cDNA formed through self-priming by 7SK RNA.

Pseudogenes having homology to small RNAs, like 7SL, 7SK, 6S, 4.5S, U1, U2, and U3 RNAs, are abundant and dispersed in the genomes of higher eukaryotes [reviewed in Weiner et al. (1986) Annu. Rev. Biochem. 55, 631-661]. To understand better the possible origin of these pseudogenes, we studied the abilities of cytoplasmic 7SL, 7SK, and nucleolar 7-2 RNAs to self-prime and result in the synthesis of cDNAs. When rat 7SK RNA was used as substrate, a 294-nucleotide-long cDNA was synthesized in vitro by reverse transcriptase, indicating that the 3' end of 7SK RNA can act in a self-priming manner to generate 7SK cDNA. When 7-2 RNA was used as a substrate, a cDNA of approximately 235 nucleotides was observed; 7SL RNA did not act as a self-primer. Earlier studies have shown that DNAs homologous to 7SK RNA are represented by a moderately reiterated family in the mammalian genomes and many of these sequences were found to be truncated 7SK pseudogenes [Murphy et al. (1984) J. Mol. Biol. 177, 575-590]. In this study, one 7SK clone from the rat genome was characterized by sequencing. This clone contained 243 base pairs homologous to the 5' end of 7SK RNA, and was flanked by direct repeats. These data suggest that, as previously proposed for some U3 pseudogenes [Bernstein et al. (1983) Cell 32, 461-472], one mechanism for the generation of truncated 7SK pseudogenes may be the integration of self-primed reverse transcripts of 7SK RNA at random genomic sites.

Animals↗
PubMed1989

Rat nucleolar 7-2 RNA is homologous to mouse mitochondrial RNase mitochondrial RNA-processing RNA.

7-2 RNA (also termed RNA M and 7SM RNA) is a noncapped small RNA present in small ribonucleoprotein particles; these particles are present in the granular compartment of the nucleolus. Some sera from patients with scleroderma specifically immunoprecipitate 7-2 RNA-containing particles (Hashimoto, C., and Steitz, J. A. (1983) J. Biol. Chem. 258, 1379-1382; Reddy, R., Tan, E. M., Henning, D., Nohga, K., and Busch, H. (1983) J. Biol. Chem. 258, 1383-1386; Reimer, G., Raska, I., Scheer, U., and Tan, E.M. (1988) Exp. Cell Res. 176, 117-128). In this study, the primary sequence of Novikoff hepatoma 7-2 RNA was determined and a possible secondary structure is presented. The Novikoff hepatoma 7-2 RNA is 94% homologous to the recently described mouse mitochondrial RNase MRP RNA, suggesting that Novikoff hepatoma 7-2 RNA may be the homologue of mouse MRP RNA. The presence of 7-2 RNA in nucleoli and in mitochondria suggests that 7-2 ribonucleoproteins, in addition to being essential components of mitochondrial RNase, may also be functional in nucleolar RNA processing and ribosome biogenesis.

Animals↗
PubMed1989

Genes for human U3 small nucleolar RNA contain highly conserved flanking sequences.

Six human genomic clones containing sequences homologous to the U3 small nuclear RNA (snRNA) were isolated and characterized. Four of these clones were real U3 snRNA genes because they were transcribed in frog oocytes and the DNA sequences corresponding to the U3 snRNA were identical to the U3 snRNA of HeLa cells. The nucleotide sequences of four true U3 snRNA genes, 537 nucleotides on the 5'-flanking region and 340 nucleotides on the 3'-flanking region, were found to be identical. In addition, the restriction patterns, upto 2 kb on the 5' side and 2.2 kb on the 3' side, appeared to be same. All the isolated U3 clones, containing 15-20 kb of genomic DNA, contained only one U3 snRNA gene, indicating that the human U3 snRNA genes are several kilobases apart. One of the U3 clones contained a full-length U3 pseudogene. Southern blot analysis of genomic DNA with cloned U3 DNA as probe indicated that human DNA contains two families of U3 genes which differ in their flanking sequences. In the 5' flanking region of human U3 snRNA genes, homology to U-gene promoter element, an octamer motif, the 'U3 box', SP1 binding sites and a consensus 3' box in the 3' flanking region, were observed. These data show that the genomic organization and the sequence motifs that control transcription of human nucleolar U3 snRNA genes are similar to those of human U1 and U2 snRNA genes and suggest common mechanism(s) in the evolution of snRNA genes.

Animals↗
PubMed1989

Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA.

U6 small nuclear RNA (snRNA), a component of eukaryotic spliceosomes, is required for splicing of nuclear pre-mRNAs. Whereas trimethylguanosine cap-containing U sn-RNAs are transcribed by RNA polymerase II, the U6 RNA is transcribed by RNA polymerase III and contains a nonnucleotide cap structure on its 5' end. We characterized the cap structure of human U6 snRNA and show that the gamma phosphate of the 5' guanosine triphosphate is methylated. The mobilities of in vivo-modified gamma phosphate from the 5' end of HeLa U6 RNA were identical to the synthetic monomethyl phosphate (CH3-O-P) in two-dimensional chromatography and two-dimensional electrophoresis. The cap structure of U6 RNA is distinct from all other cap structures characterized thus far.

Chromatography, Thin Layer↗
PubMed1989

Smooth muscle isoactin and elastin in fetal bovine lung.

The formation of elastic fiber network in the lung is developmentally regulated. In this study we first demonstrated that tropoelastin mRNA per unit total RNA in the fetal bovine lung increased from 110 to 250 days of gestation (270 day term) as measured by Northern blot analysis. To examine the extent that smooth muscle (SM) type cells contribute to this gestational increase in elastin phenotype, we utilized a dual immunofluorescent staining technique on lung sections with anti-elastin polyclonal and anti-SM isoactin monoclonal antibodies. Elastin staining was always found to localize in proximity to SM isoactin-positive cells at various stages of prenatal lung parenchymal development. Minimal, if any, elastin was seen at interstitial fibroblasts, which were negative for the SM isoactin staining. Distribution of SM (type) cells and elastic fiber together along the airways became sparse and discontinuous distally, and it seemed that formation of air sacs was between the discontinuous elastic fibers. We speculate that smooth muscle (type) cells may be the major elastogenic cells of distal airways and may play an important role in alveolar formation.

Actins↗
PubMed1989

Child abuse and neglect as seen in General Hospital, Kuala Lumpur--a two year study.

Eighty-six children diagnosed as child abuse and/or neglect were admitted to the Paediatric wards of the General Hospital, Kuala Lumpur during 1985 and 1986. Of these cases, 62 were of physical abuse, six of sexual abuse, one case of both physical and sexual abuse and 17 of neglect. There were 25 boys and 61 girls. Thirty-four of these children were Malays, 16 Chinese, 26 Indians, three mixed and seven illegal immigrants. Twenty-one were below the age of one year, 24 from one to four years, 25 from five to nine years and 16 were ten years and above. The abusers were mainly close members of the family. Of these children, 24 were sent back to their parents and 11 to their relatives home. Twenty-seven were taken into care by the Ministry of Social Welfare and the remaining seven children who were illegal immigrants, were deported with their parents. Only one child was successfully fostered. Eleven children were taken away from the hospital by their parents or guardians without the knowledge of the health staff. There were five deaths in the series.

Child↗
PubMed1988

Transcription of a U6 small nuclear RNA gene in vitro. Transcription of a mouse U6 small nuclear RNA gene in vitro by RNA polymerase III is dependent on transcription factor(s) different from transcription factors IIIA, IIIB, and IIIC.

U6 small nuclear RNA (snRNA), an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. In this study, the optimal conditions for transcription of the U6 snRNA gene in vitro were found to be similar to conditions optimal for transcription of 5S RNA genes. To purify the trans-acting factors necessary for the transcription of the U6 RNA gene, HeLa cell extracts were fractionated on a DEAE-Sephadex column, and three fractions, designated DE-50, DE-175, and DE-500, were obtained by stepwise elution with 50, 175, and 500 mM ammonium sulfate, respectively. DE-175 fraction transcribed tRNA and 5S RNA genes but not a mouse U6 RNA gene. Complementation of the DE-175 fraction with the DE-50 fraction resulted in the transcription of the U6 RNA gene. Experiments in which the transcription factor (TFIIIA) was selectively inactivated indicated that TFIIIA is not required for the transcription of the U6 RNA gene. These results show that the U6 snRNA gene, although transcribed by RNA polymerase III, differs from tRNA and 5S RNA genes in that factors other than TFIIIA, -IIIB, and -IIIC are required for U6 gene transcription in vitro.

Cloning, Molecular↗
PubMed1988

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

Whereas the genes coding for trimethyl guanosine-capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis-regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5' deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5' distal sequence; these studies revealed that the distal element acts as an orientation-dependent enhancer when present upstream to the gene, while it is orientation-independent but distance-dependent enhancer when placed down-stream to the U6 gene. Analysis of 3' deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3' end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III.

Animals↗
PubMed1988

Characterization of Novikoff hepatoma small RNAs homologous to repetitive DNAs.

Three minor small RNA species from Novikoff hepatoma cells, with homology to repetitive DNA sequences, have been identified and characterized. These small RNAs, designated 5.1S, 6S and T3 RNAs, show homology to Alu 1, Alu 2, and Alu 3 sequences, respectively. 6S and T3 RNAs were found both in the nucleus and cytoplasm, whereas 5.1S RNA was not found in the nucleus. Neural tissues were found to contain a 6S-sized BC1 RNA with homology to I.D. sequences; in contrast, the current study shows that Novikoff hepatoma cells contain a 75-80 nucleotide long (T3) RNA, homologous to I.D. sequences. These data suggest that BC1 and T3 small RNAs, homologous to I.D. sequences, are expressed in a tissue-specific manner. These results also show that in addition to the abundant 7SL, 4.5S and 4.5S1 RNAs having homology to repetitive DNA, Novikoff hepatoma cells also contain several minor small RNAs with homology to repetitive sequences.

Animals↗