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Biomedical subjects

R A Padgett

Publications and source records attributed to R A Padgett.

35 records · Page 2Linked to original sources
PubMed1985

Trans splicing of mRNA precursors in vitro.

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.

Adenosine Triphosphate↗
PubMed1985

Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary.

Animals↗
PubMed1985

Bioluminescence screening for bacteriuria.

A rapid screening test (45 min) for bacteriuria was evaluated in 1,000 clinical urine specimens. The test procedure is based upon firefly luciferase analysis of bacterial ATP and uses the Lumac kit and Lumac M2010 Biocounter (3M Co., St. Paul, Minn.). The procedure allows for removal and destruction of nonbacterial ATP and subsequent analysis of bacterial ATP by firefly luciferase with a single photon counter. Results, expressed in relative light units, were compared with actual CFU by the calibrated loop technique. Sensitivities and specificities were calculated separately for clean-catch midstream specimens and for urines obtained by catheterization. The sensitivity for 719 clean-catch specimens with a Lumac cutoff of greater than or equal to 500 relative light units, representing greater than or equal to 10(5) CFU/ml, was 93%. The sensitivity for 281 catheterized specimens with a Lumac cutoff of greater than or equal to 200 relative light units, representing greater than or equal to 10(4) CFU/ml, was 95%. There were 19 false-negative results in the 1,000 specimens tested; more than 50% of these were contaminated cultures and were not considered significant in determining bacteriuria. In conclusion, the Lumac bioluminescence assay is a reliable, rapid bacteriuria screening technique with the potential of reducing the laboratory cost and for reducing the turnaround time in processing negative urine cultures.

Bacteriological Techniques↗
PubMed1984

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

Adenoviruses, Human↗
PubMed1984

Recognition of cap structure in splicing in vitro of mRNA precursors.

Substrate RNAs are only efficiently spliced in HeLa whole-cell extract when they possess capped 5' termini. This cap requirement is observed with substrate RNAs prepared by transcription with either mammalian RNA polymerase II or bacterial RNA polymerase. Addition of less than 10 microM of cap analogs such as m7G(5')ppp(5')N or m7GTP strongly inhibits splicing of capped RNAs. This observation, as well as experiments following the fate of substrate RNA, indicates that the dependence of splicing on a cap structure is not due to an effect on RNA stability. More interestingly, cap analogs inhibit splicing when added at the start of the reaction but not at later times of incubation. This suggests that the cap recognition might be an important step in the formation of a specific ribonucleoprotein complex required for splicing.

DNA-Directed RNA Polymerases↗
PubMed1984

Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate.

Four RNA products have been characterized from a soluble system that accurately splices purified substrate RNA. The labeled substrate RNA contained the first and second exons of the major late transcription unit of adenovirus 2 and a shortened form of the first intervening sequence. One of these RNA products was the excised intervening sequence which accumulated quantitatively with the level of splicing. This RNA species has an unusual structure and is modified at internal sites. A potential intermediate in the splicing pathway which is cleaved at the 5' splice site of the first exon has been isolated. This RNA species is also modified at sites within the first intervening sequence. These results suggest that the splicing of mRNA precursors may involve sites in the intervening sequence, cleavage at the 5' splice site, cleavage at the 3' splice site, and ligation of the two exons.

Adenoviruses, Human↗
PubMed1983

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein.

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

Adenoviruses, Human↗
PubMed1983

Splicing of adenovirus RNA in a cell-free transcription system.

A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

Adenoviruses, Human↗
PubMed1982

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

Amidohydrolases↗
PubMed1982

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.

Animals↗
PubMed1982

Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

Animals↗
PubMed1979

Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells.

Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.

Animals↗
PubMed1979

N-(Phosphonacetyl)-L-aspartate-resistant hamster cells overaccumulate a single mRNA coding for the multifunctional protein that catalyzes the first steps of UMP synthesis.

We have investigated the mechanism of overproduction of the multifunctional protein catalyzing the first three steps of UMP biosynthesis in stable mutants of Syrian hamster cells in culture. The rate of degradation of this protein is unaltered in one mutant cell line which overproduces it by 118-fold. In all mutants tested, the increase in the rate of synthesis of this protein is equal to the increase in its steady state concentration. There is a similar correlation between steady state levels of this protein in vivo and the capacity of polysomal RNA isolated from these cells to direct the synthesis of the protein in vitro. The one mutant cell line studied contains large amounts of a polysomal poly(A)-containing RNA (Mr = 2.7 +/- 0.2 times 10(6)) that is not detected in wild type cells. This large RNA co-sediments in sucrose gradients with the capacity to direct the synthesis of the multifunctional protein in vitro.

Animals↗
PubMed1978

Herpes simplex virus necleic acid synthesis following infection of non-permissive XC cells.

DNA hybridization kinetic analysis of cellular DNA following high multiplicity infection of non-permissive XC cells by herpes simplex virus type I showed that HSV DNA penetrates to the nucleus of the cell but that the number of virus DNA copies present in each cell quickly begins to decline. There did not appear to be any net virus DNA synthesis and the loss of virus DNA copies continued until there was approximately one per haploid genome equivalent. HSV-2 likewise did not show any detectable virus DNA replication. The residual virus information was stable for more than 48 h. CsCl density gradient analysis of the infected cell DNA suggested an association between the HSV DNA and that of the cells. Network analysis also supported the suggestion that a stable association between the virus DNA and host DNA begins shortly after infection. Cell division resulted in the segregation of the virus DNA but not its loss from the cell population. Virus-specific RNA synthesis was easily detectable and 40 to 50% of a labelled DNA probe was converted to an RNA:DNA hybrid.

Cell Line↗
PubMedDate not supplied

Cofactor requirements of splicing of purified messenger RNA precursors.

The origin and functions of introns in protein coding genes is one of the enigmas of molecular biology. Splicing processes that remove intervening sequences from precursor RNAs must have either predated or co-evolved with introns. Inferences about the origin of introns and the possible modes of regulation of splicing should emerge from an understanding of the biochemical mechanisms of splicing. The biochemistry of splicing of tRNA and rRNA precursors has rapidly advanced with the development of in vitro reactions containing soluble components that duplicate in vivo reactions. We have recently shown that accurate splicing of an adenovirus mRNA precursor occurs during a coupled transcription/splicing reaction in a soluble whole cell extract. We now report that an exogenous RNA substrate containing the first and second leaders of adenovirus 2 is accurately spliced when added to an extract of HeLa cells. ATP and Mg2+ are essential cofactors for the reaction. The time course of splicing is unusual; a lag of 45 min is observed before the appearance of splicing product.

Adenosine Triphosphate↗