Regulatory functions of 3'UTRs.
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3' Untranslated Regions↗
Biomedical subjects
J A Siedlecki
Publications and source records attributed to J A Siedlecki.
Detection of circulating melanoma cells in peripheral blood by a two-marker RT-PCR assay.
The aim of this study was to develop a highly sensitive two-marker assay for the detection of circulating melanoma cells in patients' blood using a reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed the usefulness of two different sets of markers: tyrosinase and MUC-18 (TYR/MUC-18), and tyrosinase and MART 1 (TYR/MART 1). Total cellular RNA was isolated from 337 blood samples from 80 melanoma patients at different stages of the disease. All patients had undergone primary surgery. Assay sensitivity and specificity were confirmed using three different melanoma cell lines and two different fibroblast lines. In addition, blood from 47 healthy subjects and 10 patients with non-melanoma cancer was used as a negative control. We found that two-marker analysis is more accurate than the single tyrosinase assay. The frequency of melanoma cell detection in patients' blood was about 10% higher when the TYR/MART 1 two-marker assay was used. Using this assay we did not find any statistical correlation between the molecular markers and the UICC stage of disease or the Breslow thickness or Clark level of the primary melanoma. The frequency of melanoma cell detection with the TYR/MUC-18 two-marker assay was even higher than the TYR/MART 1 assay, but unfortunately the MUC-18 transcript was also present in about 20% of healthy subjects. Therefore we do not recommend the use of MUC-18 as a standard value marker.
Adult↗
Alternative forms of beta-pol mRNA are not tumor-specific and are not the result of mutations in the DNA.
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Alternative Splicing↗
Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.
Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.
Alternative Splicing↗
Alternative splicing of DNA polymerase beta mRNA is not tumor-specific.
Mammalian DNA polymerase beta is a crucial enzyme in cell genomic maintenance. Its structure is highly conserved. Some splice variants of beta-pol mRNA were observed. One alternative splice DNA polymerase beta mRNA, generated by 87 nt deletion (exon 11) in the catalytic domain of this enzyme, was suggested to be responsible for genomic instability in tumorigenesis and in genetic disorder (Werner syndrome). Here, we show that exon-11-deleted beta-pol mRNA is present in all examined normal and tumor tissues as well as in resting or PHA-stimulated peripheral-blood mononuclear cells. This finding proves that the presence of the exon-11 alternative splicing variant of beta-pol mRNA is not tumor-specific.
Alternative Splicing↗
Cloning and characterization of the 5' part of the high molecular weight transcript of rat DNA polymerase beta.
A rat brain cDNA library has been constructed. Three identical clones of about 2.2 kb representing high molecular weight DNA polymerase beta transcript were found. Sequencing proved our earlier suggestion that both high and low molecular weight DNA polymerase beta transcripts have the same open reading frame and differ mainly at the 3' end. Because of that, alternative polyadenylation is discussed as a possible mechanism for tissue- and development-specific regulation of the DNA polymerase beta gene expression.
Animals↗
[Myelodysplastic syndrome associated with clonal proliferation of B lymphocytes].
We present an unusual case of the myelodysplastic syndrome (subtype refractory anemia with the excess of blasts in transformation--RAEB-t) associated with significant increase of IgG (4,700 mg/dl), lambda (160 U/dl) in blood serum and circulating clone of B lymphocytes SIgG, lambda, manifesting clonal rearrangement of JH domain. Peripheral blood cells of the patient showed two different chromosomal abnormalities: 47,XY, + del/8/p? and 47,XY, +22, +14, -19. We suppose that two independent neoplastic clones are developed in the described case, i.e. a population displaying markers of myeloblasts and monoblasts, and a clone of B lymphocytes.
Aged↗
Effect of busulphan treatment and elevated temperature on the expression of the beta-pol gene in rat testis.
Changes in the expression pattern of the DNA polymerase beta gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of beta-pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of beta-pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptorchidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of beta-pol mRNA. Involvement of DNA polymerase beta in meiotic recombination is discussed.
Animals↗
Changes in the DNA polymerase beta gene expression during development of lung, brain, and testis suggest an involvement of the enzyme in DNA recombination.
Changes in the expression pattern of DNA polymerase beta gene during rat lung, brain, and testis development have been investigated. A decrease in the level of beta-pol mRNA was observed during postnatal development of lung and brain. By contrast, an almost 20-fold increase in the level of beta-pol mRNA was observed during spermatogenesis. For most adult rat tissues the abundance of beta-pol mRNA was low compared with that of beta-actin mRNA. Northern blot analysis revealed four distinct transcripts hybridizing to beta-pol probes. At least two of them, 1.4 kb and 4.0 kb, were products of a beta-polymerase gene. The changes in the expression pattern during lung and brain development, and during spermatogenesis, suggest involvement of DNA polymerase beta in gap-filling DNA synthesis during recombination.
Animals↗
Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.
A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed.
Animals↗
Proto-oncogene c-fos induction in rat hippocampus.
The apparently non-specific accumulation of c-fos proto-oncogene mRNA was found in rat hippocampus as a result of injection of either glutamate, noradrenaline, or physiological saline. There was no c-fos gene induction following an electroconvulsive shock.
Animals↗
[DNA polymerases in the cells of higher eukaryotes].
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Animals↗
The ability of DNA polymerase beta to synthesize DNA beyond the gap with displacement of the non-replicated strand.
Study was made of the ability of calf thymus DNA polymerases alpha and beta to replicate templates containing a small gap. It was found that during extensive replication of activated DNA or synthetic template.primers or specially prepared circular DNA containing a small gap, catalyzed by DNA polymerase alpha, the levels of incorporated nucleotides corresponded to the amounts of the single-stranded fraction of these templates. In contrast, in the reaction catalyzed by DNA polymerase beta the amounts of products were several times greater. The ability to synthesize the product in a great excess was a specific feature of the latter enzyme. An analysis of the gap-filling products by sucrose gradient centrifugation, gel electrophoresis and Southern hybridization showed that, contrary to DNA polymerase alpha, DNA polymerase beta exhibited the ability to synthesize DNA not only within but also beyond the gap. The "net" DNA product is complementary to the template strand. It is suggested that DNA was synthesized beyond the gap by displacement of the non-replicated strand.
Animals↗
DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides.
A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.
Cell Nucleus↗
Reactions of calf thymus DNA polymerases alpha and beta with native DNA damaged by thymine starvation or by methyl methanesulphonate treatment with Escherichia coli cells.
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Animals↗
Net DNA synthesis catalysed by calf thymus DNA polymerase beta.
1. The amounts of deoxynucleotides incorporated during an extensive replication by DNA polymerase alpha into poly(dA)-oligo(dT)12-18 and damaged DNA containing 2.5 incisions per molecule and 3.7% single-stranded DNA, corresponded to the amounts of non-complexed poly(dA) and single-stranded fraction of DNA, respectively. The amounts of the corresponding DNA polymerase beta products were several times higher. In the case of activated DNA they exceeded input DNA. The DNA polymerase beta reaction on this template was continued till substrate exhaustion. 2. The reaction of DNA polymerase beta with activated DNA, leading to net DNA synthesis, was template-directed, required Mg2+ and four deoxynucleoside triphosphates; was not inhibited by DNA polymerase alpha inhibitors, but was sensitive to 2',3'-dideoxythymidine triphosphate. The DNA product was completely digestable by DNAse I and showed a base ratio typical of calf thymus DNA. 3. The essential difference in the reaction mechanism between DNA polymerase alpha and beta suggests the ability of the latter enzyme to synthesize DNA with displacement of the non-replicated strand.
Animals↗
Evidence implying DNA polymerase beta function in excision repair.
Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, being, as expected, 40 times higher than for T-DNA. Likewise, the differences in the yield of the s.s.act.DNA or s.s.BU-DNA replication between both enzymes were negligible. In contrast, damaged native DNA was 6 - 30 times more extensively replicated by DNA polymerase beta than alpha. We propose that this is due to the greater ability of DNA polymerase beta compared with alpha to replicate single-stranded gaps, the presence of which is more likely in damaged BU-DNA than in T-DNA and act.DNA.
Animals↗