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Biomedical subjects

R G Pergolizzi

Publications and source records attributed to R G Pergolizzi.

29 records · Page 2Linked to original sources
PubMed1983

Abnormal splice in a mutant human beta-globin gene not at the site of a mutation.

We have studied the expression of a cloned mutant human beta-globin gene in tissue culture cells. The gene, which was previously isolated from the chromosomal DNA of an individual with a low level of normal beta-globin expression (beta+-thalassemia), contains five mutations inside the large intervening sequence (IVS2), as well as a silent change in codon 2. This beta-thalassemia gene (thal) was inserted into a plasmid that is replicated and transcribed in a line of monkey kidney cells in culture. S1 nuclease mapping of the beta-globin RNA transcribed from this gene indicates that some of the beta-globin RNA is spliced abnormally by using a cryptic 3' splice sequence normally present in IVS2 but not used in processing the normal beta-globin transcript. The cryptic 3' splice site is not the site of a mutation in the thal gene. Because neither the 5' or 3' splice junction nor the cryptic site is mutated in this gene, it is most likely that the mutation at position 705 of IVS2, the only nonpolymorphic change in the gene, interferes indirectly with normal processing. These results suggest that certain sequences within IVS must be conserved to prevent abnormal splicing and loss of gene function.

Base Sequence↗
PubMed1982

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.

Base Sequence↗
PubMed1980

The effect of exogenous nutrients on the biosynthesis of Y base in tRNAPHe from Ehrlich ascites carcinoma.

In contrast to normal eukaryotic tissue, which contains a single isoaccepting species of tRNAPhe, the Ehrlich ascites carcinoma contains a spectrum of tRNAPhe species varying in their relative degree of posttranscriptional modification. The occurrence of multiple species involves an incomplete biosynthesis of the Y base, a complex hypermodified nucleoside located adjacent to the 3' end of the anticodon. Following intraperitoneal innoculation of a mixture of essential amino acids and vitamins into these mice, the majority of the tumor tRNAPhe behaved like the single liver tRNAPhe species. This suggests that under these conditions the modification of Y base was completed.

Amino Acids, Essential↗
PubMed1979

Synthesis and coding properties of dinucleoside diphosphates containing alky pyrimidines which are formed by the action of carcinogens on nucleic acids.

Dinucleoside diphosphates of the general type pGpN have been prepared enzymatically using ribonuclease N1. Alkylated uridines or cytidines, which are products of carcinogens acting on nucleic acids, were tested in dinucleoside diphosphates for their ability to stimulate the binding of Ala- or Val-tRNA to ribosomes. O2-Ethyl C and 3-methyl C functioned as U, but not as C. In contrast, 3-methyl U behaved as C, but not as U. Both O2 and O4-ethyl U could be recognized as C or U, although binding in both cases was weak. Thus, modifications of the hydrogen-bonding sites of U or C causes miscoding and could be considered to represent mutagenic reactions.

Codon↗
PubMed1979

Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells.

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.

Animals↗
PubMed1978

Formation of phenylalanine transfer RNA lacking the wye base in Vero cells during methionine starvation.

Vero, a cell line derived from African green monkey kidney, normally contains a single species of tRNAPhe (tRNA2Phe), containing a hypermodified base, wye (originally called Y), next to the 3' end of the anticodon. When methionine is removed from the growth medium, there appears a new tRNAPhe species (tRNA1Phe) lacking the wye base and eluting early from reversed phase chromatography columns. Its appearance is not due to the cessation of cell growth. Addition of methionine to cells containing both species of tRNAPhe leads to the disappearance of tRNA1Phe. When [methyl-3H5methionine is added in the presence of actinomycin D, which blocks new RNA synthesis, label appears in the wye base of tRNA2Phe. These results are consistent with the model that tRNA1Phe is an undermodifed precursor of tRNA2Phe and that methionine is required for modification to the mature form.

Cell Line↗
PubMedDate not supplied

Polymerase chain reaction analysis of fragile X mutations.

The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.

Base Sequence↗
PubMedDate not supplied

Analysis of chromosome 22 loci in meningioma. Alterations in the leukemia inhibitory factor (LIF) locus.

Meningiomas are typically benign tumors arising from arachnoidal cells at the base of the brain. Meningioma is thought to result from the loss or inactivation of a putative tumor suppressor gene located on chromosome 22. We analyzed a set of meningioma DNA specimens by Southern blot hybridization with chromosome 22-specific probes and by PCR using oligomer primers and probes specific to the leukemia inhibitory factor (LIF) gene. Southern analysis suggested that a subset of our specimens are monosomic for 22q11-qter and may have lost one entire copy of chromosome 22. The gene(s) involved in the etiology of meningioma has been localized to 22q11.2-12.3. The locus encoding LIF, a factor that affects the differentiation and proliferation of numerous cell types, has also been localized to this region, at 22q12.1-12.2. The partial overlap of these loci, coupled with the known involvement of the LIF gene product in growth and differentiation, suggested that the LIF locus may be associated with the meningioma defect. We have examined the LIF locus in meningioma specimens at the molecular level by PCR, and by DNA and RNA gel-blot hybridizations. Alterations in the structure and/or expression of the LIF locus were detected in several specimens, including the subset that were shown to be monosomic for 22q. All of our tumor specimens were shown to be undermethylated at the LIF locus relative to constitutional DNA from the same patients. Sequence analysis of LIF cDNA from a meningioma revealed the existence of a novel, alternatively spliced LIF mRNA. These results suggest that the LIF gene may be near the putative tumor suppressor locus associated with the development of this phenotype.

Adult↗
PubMedDate not supplied

A fifth human cytomegalovirus glycoprotein B genotype.

Genetic variation in glycoprotein B (gB) may play a role in human cytomegalovirus (HCMV) pathogenesis. Using restriction endonuclease digestion and DNA sequencing, a unique gB genotype was identified in eight HCMV strains isolated from five patients with the acquired immune deficiency syndrome. Nucleic acid homology to the four previously described gB genotypes ranged from 79 to 91% for the two major variable regions of gB. Studies of the role of gB in HCMV pathogenesis should recognize the existence of live gB genotypes.

AIDS-Related Opportunistic Infections↗