Does 77C-->G in PTPRC modify autoimmune disorders linked to the major histocompatibility locus?

A 77G allele of the gene encoding CD45, also known as the protein tyrosine phosphatase receptor-type C gene (PTPRC), has been associated with multiple sclerosis (MS). Here we determine allele frequencies in large numbers of MS patients, primary immunodeficiencies linked to major histocompatibility complex (MHC) locus and over 1,000 controls to assess whether aberrant splicing of PTPRC caused by the 77C-->G polymorphism results in increased susceptibility to these diseases. Our results show no difference in the frequency of the 77G allele in patients and controls and thus do not support a causative role for the polymorphism in the development of disorders with a strong autoimmune component in etiology.

Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes.

The S. cerevisiae RAD51 gene product exerts important functions in meiotic and mitotic recombination, as well as in the repair of DNA double-strand breaks. We have studied the expression of the human RAD51 (HsRAD51) gene in primary human peripheral blood lymphocytes (PBLs). The HsRAD51 mRNA level increased three fold in mitogen stimulated PBLs, with a peak in the late S phase. A five fold increase of HsRAD51 protein levels was observed in late G2. Specific inhibition of DNA synthesis with aphidicolin did not block the induction of the HsRAD51 protein, indicating that HsRAD51 expression is independent of DNA replication. In contrast, after inhibition of RNA synthesis with actinomycin D and protein synthesis with cycloheximide, the HsRAD51 protein level decreased rapidly. Taken together, these results indicate that the HsRAD51 gene is transcriptionally regulated in human PBLs, and exerts its function during the late S and G2 phases of the cell cycle.

Missense mutations and evolutionary conserved amino acids at the human hypoxanthine phosphoribosyl-transferase locus.

Molecular characterization of in vivo mutation at the human hypoxanthine phosphoribosyltransferase (hprt) locus has revealed a broad spectrum of mutation, both with regard to germ-line mutation in Lesch-Nyhan and gout patients, and somatic mutation in 6-thioguanine resistant T-lymphocytes from healthy individuals. The pattern of missense mutation shows a non-random distribution with a preferential location to codons for amino acids which are identical in human and the two parasites Schistosoma mansoni and Plasmodium falciparum. Although these 'evolutionary conserved' amino acids account for only 32% of the amino acids in the human hprt protein, they are involved in 76% of the missense mutations at the hprt locus in human T-lymphocytes, 67% in Lesch-Nyhan patients (with severe hprt-deficiency), but only 43% in gout patients (with partial hprt deficiency). This observation supports the notion that evolutionary conserved amino acids constitute functionally important sites in the hprt enzyme, and missense mutations affecting these amino acids will often lead to complete loss of enzyme activity. Substitutions of 'non-conserved' amino acids cause less severe hprt-deficiency (as seen in the gout patients), or may even escape clinical diagnosis. These considerations are important for the understanding of structure-activity relationships in the hprt protein, possible differences between hprt mutational spectra in germ-line and somatic cells, and the mutational spectra induced by specific exogeneous mutagens.

p53 splice acceptor site mutation and increased HsRAD51 protein expression in Bloom's syndrome GM1492 fibroblasts.

GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.

Proteolytic cleavage of HsRad51 during apoptosis.

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Alzheimer's disease: molecular genetics and transgenic animal models.

Disease-causing mutations in the amyloid precursor protein (APP) gene have been found on chromosome 21 during the last 2 years in some early onset Alzheimer's disease (AD) families. Genetic evidence shows that other genes than the APP are also involved in the aetiology of AD. Linkage to a loci on chromosome 14 has been found in early onset disease. The identification of APP mutation has led to the realization that APP mismetabolism is a central event in the aetiology and pathogenesis of the disease. Experiments to test this in transgenic mice have so far met with little success. There are many possible explanations for the problems to generate transgenic mice. These include the possibilities that mice are incapable of developing AD for reasons dependent on their APP sequence; and that appropriate regulation of APP gene is required for pathology to develop. Current attempts that seem promising to model the disease pathology are the use of homologous recombination to insert the pathogenic mutation and transfection of YACs into transgenic animals.

Frequency and cell specificity of T-cell receptor interlocus recombination in human cells.

Immunoglobulin and T-cell receptor (TCR) genes are assembled by a site-specific rearrangement known as V(D)J [variable-(diversity)-joining] recombination. These rearrangements occur normally in pre-B- and pre-T-cells using signal sequences adjacent to coding exons for immunoglobulin and TCR genes, respectively. However, aberrant recombination may result in the generation of hybrid TCR genes by joining of TCR-beta with TCR-gamma specific sequences. Such hybrid TCR genes occur at a low frequency in peripheral blood lymphocytes (PBL) of healthy individuals, and can be detected by PCR amplification. We have determined the in vivo frequency of hybrid V gamma-J beta 1 TCR (hybrid TCR) genes in lymphocyte DNA from 12 healthy individuals. The average frequency was found to be 5.83 in 0.75 x 10(6) PBL, with a threefold difference between the highest and lowest individual value. The presence of similar TCR gene rearrangements in individual samples suggests that T-cells with a hybrid TCR gene are capable of clonal expansion in vivo. The individual hybrid TCR gene frequency remained relatively constant during 72 hours of in vitro cultivation. In long-term culture, the frequency gradually decreased, and after 28 days no hybrid TCR genes were detectable in lymphocyte DNA. These results show that T-cells with a hybrid TCR gene are able to respond to mitogen stimulation in vitro, and may have a proliferative disadvantage or are selected against during prolonged in vitro cultivation. No hybrid TCR genes were detected in ten proliferating T-cell clones, indicating that the rate of hybrid TCR gene formation is < 2.0 x 10(-8) per cell per cell division. No hybrid TCR genes were detected in DNA from B-lymphocytes, sperm, granulocytes, fibroblasts, keratinocytes, and three B-lymphoblastoid ataxia telangiectasia cell lines. In agreement with previous reports, the frequency of hybrid TCR genes in peripheral blood DNA from two ataxia telangiectasia patients was found to be more than 15-fold higher than in lymphocytes from normal individuals. These data show that formation of hybrid TCR genes is restricted to T-cells in vivo, and occurs at a very low frequency, if at all, in proliferating T-cells in vitro, and with an increased frequency in patients with ataxia telangiectasia.

Variations in the frequency of T-cell receptor beta/gamma-interlocus recombination in long-term cultures of non-irradiated and X- and gamma-irradiated human lymphocytes.

PURPOSE: The increased level of illegitimate V(D)J recombination at the T-cell receptor (TCR) loci in lymphoid tumours as well as in T lymphocytes of ataxia telangiectasia patients and humans exposed to carcinogens in vivo suggest that site-specific interlocus recombination events could serve as markers of genomic instability and early genetic changes associated with carcinogenesis. The purpose of this study was to investigate the ability of ionizing radiation to induce TCRbeta/gamma-interlocus rearrangements in human lymphocytes in vitro. MATERIALS AND METHODS: Peripheral blood lymphocytes (PBL) from two healthy donors were exposed to 3 Gy of either X- or gamma-irradiation in vitro. Growth factor-stimulated cell cultures were established, and cell samples for DNA extraction were taken immediately after exposure and at several time points during long-term growth. A PCR-based method was used to measure the frequency of variant cells with Vgamma-Jbeta1 TCR rearrangements. RESULTS: The frequency of TCRbeta/gamma-variant cells was not significantly different in the irradiated and control cultures at any time studied up to 55 days after PHA-stimulation, indicatin that V(D)J-mediated Vgamma-Jbeta1 rearrangement is not induced by X- or gamma-irradiation under these conditions. However, in both irradiated and non-irradiated cultures, the frequency of TCRbeta/gamma variants increased approximately fourfold after mitogen stimulation, from a normal background level of 0.3-0.4 x 10(-5) to 1.3-1.6 x 10(-5) at days 4-9. These levels then gradually declined during prolonged cultivation, and after 2-4 weeks the frequency of variant cells was below the detection limit ( < 0.13 x 10(-5)). CONCLUSIONS: These results provide no evidence that TCRbeta/gamma gene rearrangements can be induced by X- or gamma-irradiation in vitro. However, in contrast with cells with normal TCR receptors, TCRbeta/gamma-variant cells display a relative growth advantage for 1-2 weeks, followed by gradual loss of proliferative capacity. Eventually, they are eliminated from the cell population or outnumbered by cells with normal TCR. If there are similar differences in vivo between cells with hybrid and normal TCR, this may explain the previously reported time- and season-dependent changes in the frequency of cells with hybrid TCR in occupationally exposed populations and individuals receiving cytostatic treatment.

Mutagen-induced recombination in mammalian cells in vitro.

It is now clear from in vitro studies that mutagens induce recombination in the cell, both homologous and nonhomologous exchanges. The recombination events induced are extrachromosomal events, exchanges between extrachromosomal DNA and chromosomes, and inter- as well as intrachromosomal exchanges. However, not all types of DNA damage can induce recombination. The mechanisms involved in the induction process are not known but may involve activation of DNA repair systems. In addition, stimulation of mRNA transcription by mutagens, different recombination pathways and how the assay system is constructed may affect the frequency and characteristics of the observed recombination events.

Mechanisms for recombination between stably integrated vector sequences in CHO cells.

Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intrachromatidal deletion and gene conversion. DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene fragments together. One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells.

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.

Mutagen-induced recombination between stably integrated neo gene fragments in CHO and EM9 cells.

The ability of mutagenic agents to induce homologous recombination was studied in a 'normal' Chinese hamster ovary (CHO) cell clone (CHO:5) and a cell clone (EM9:2) derived from the presumptive DNA repair-deficient mutant cell line EM9, which has a high spontaneous sister-chromatid exchange (SCE) level and shows hypersensitivity towards monofunctional alkylating agents and bromodeoxyuridine (BrdUrd). The 2 cell clones have been transfected with and allowed to incorporate stable genomic inserts of the vector pIII-14gpt, which contains 2 tandemly arranged neo gene fragments with a common 400-bp region of homology. Recombination between the truncated neo genes gives rise to geneticin sulfate (G 418)-resistant revertants with a spontaneous frequency of about 10(-4) in both cell clones. In CHO:5 an increased frequency of revertants was obtained after treatment with methyl methanesulfonate (MMS) and mitomycin C (MMC), while HN2, benz[a]pyrene diolepoxide (BPDE) and X-irradiation gave negative results. EM9:2 showed about the same increase of revertants after treatment with MMS as CHO:5, but in a 10-fold lower dose range. HN2 as well as BrdUrd induced revertants in EM9:2. These results show that mutagenic agents (MMS, MMC, HN2, BrdUrd) can induce homologous recombination in this system. This effect does not seem to be an unspecific effect of DNA damage (no effect of X-ray and BPDE), or related to SCE induction in general (similar spontaneous and MMS-induced frequencies of revertants in CHO:5 and EM9:2). However, the positive effect of BrdUrd in EM9:2 and the difference between CHO:5 (negative) and EM9:2 (positive) with regard to HN2-induced revertants suggest that certain types of DNA damage are more recombinogenic in EM9 than in 'normal' CHO cells, which possibly reflects the specific mutation in the former cell line.

Effects of UV-irradiation on the SCE frequency in human lymphocyte cultures.

UV-irradiation (254 nm) was found to induce a smaller increase of SCE in human lymphocytes than in human fibroblasts and CHO cells. The UV-induced SCE frequency in human lymphocytes was not influenced by the duration between irradiation and the subsequent S-phase. UV-irradiated lymphocytes showed a slightly more than additive response to the SCE-inducing effect of HN2 and acetaldehyde in comparison with non-irradiated cells. The UV-induced SCE frequency was similar in lymphocyte cultures containing 20 and 100 microM of BrdUrd. The results suggest that human lymphocytes are relatively insensitive to the SCE-inducing effect of UV-irradiation, and that SCE-inducing damage caused by UV is not removed during the G1 phase in these cells.

Unequal SCE is a rare event in homologous recombination between duplicated neo gene fragments in CHO cells.

The frequency of sister-chromatid exchange (SCE) was studied in Chinese hamster ovary (CHO) cell lines with stable insertions of the vector pIII-14gpt which contains 2 truncated neomycin resistance (neo) gene fragments. Recombination between regions of homology in the 2 fragments can restore a functional neo gene and make the cell resistant to the antibiotic G418, a neomycin analogue. Unequal SCE would be one of several possible mechanisms for this event. The observed spontaneous rate of formation of G418-resistant subclones was approximately 6.4 x 10(-6) per cell per generation, as compared to the estimated spontaneous frequency of 3 SCE per cell per generation. Given this SCE frequency, the probability of an SCE occurring in a target site of about 1600 bp (the distance separating the homologous regions in the neo fragments) would be about 8 x 10(-7) per cell per generation, or approximately one tenth of the estimated rate of recombination. Treatment of the cells with methyl methanesulfonate (MMS, 50 x 10(-6) M) induced about 80-90 SCE per cell, corresponding to a probability of 2 x 10(-5) SCE per 1600-bp target per cell. In the same cell culture, MMS treatment induced 4-8 x 10(-4) recombination events per cell giving rise to G418 resistance. Cells treated with HN2 (up to 4 x 10(-6) M) showed a significant increase in SCEs, but no change in the frequency of G418-resistant revertants. These results suggest that the 2 pathways leading to SCE and recombination respectively are uncoupled, and only a small fraction of the recombination events, if any, are due to unequal SCE in this system.

Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes.

The induction of SCE was studied in PHA-stimulated human lymphocytes exposed to nitrogen mustard (HN2) or methyl methanesulfonate (MMS) for various time periods in the G1 phase. HN2 was found to induce about 10 times more SCE when cells were exposed in late G1 (24 h after PHA) as compared to early G1 (immediately after PHA). In contrast, only a small difference was observed between cells exposed to MMS in late or early G1. The results suggest that different types of SCE-inducing alkylating damage agents are removed at widely different rates in human G1-lymphocytes.

The frequency of illegitimate TCRbeta/gamma gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation.

Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.