Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein–Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. The XLP gene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions. We describe here the presence of small deletions and intragenic mutations that specifically disrupt a gene named DSHP in 6 of 10 unrelated patients with XLP. This gene encodes a predicted protein of 128 amino acids composing a single SH2 domain with extensive homology to the SH2 domain of SHIP, an inositol polyphosphate 5-phosphatase that functions as a negative regulator of lymphocyte activation. DSHP is expressed in transformed T cell lines and is induced following in vitro activation of peripheral blood T lymphocytes. Expression of DSHP is restricted in vivo to lymphoid tissues, and RNA in situ hybridization demonstrates DSHP expression in activated T and B cell regions of reactive lymph nodes and in both T and B cell neoplasms. These observations confirm the identity of DSHP as the gene responsible for XLP, and suggest a role in the regulation of lymphocyte activation and proliferation. Induction of DSHP may sustain the immune response by interfering with SHIP-mediated inhibition of lymphocyte activation, while its inactivation in XLP patients results in a selective immunodeficiency to EBV.

Type 2 diabetes: Evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, x̂ = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (x̂ = 57 cM, recurrence risk, λ̂s = 1.25, P = 0.009). Weighted logarithm of odds scores of 2.00 (x̂ = 69.5 cM, P = 0.010) and 1.92 (x̂ = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2.12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4α) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Y chromosome polymorphism is a strong determinant of male fitness in Drosophila melanogaster

In many species, the Y (or W) chromosome carries relatively few functional genes. This observation motivates the null hypothesis that the Y will be a minor contributor to genetic variation for fitness. Previous data and theory supported the null hypothesis, but evidence presented here shows that the Y of Drosophila melanogaster is a major determinant of a male's total fitness, with standing genetic variation estimated to be 68% of that of an entire X/autosome genomic haplotype. Most Y-linked genes are expressed during spermatogenesis, and correspondingly, we found that the Y influences fitness primarily through its effect on a male's reproductive success (sperm competition and/or mating success) rather than his egg-to-adult viability. But the fitness of a Y highly depended on the genetic makeup of its bearer, reverting from high to low in different genetic backgrounds. This pattern leads to large epistatic (inconsistent among backgrounds) but no additive (consistent among backgrounds) Y-linked genetic variance for fitness. On a microevolutionary scale, the observed large epistatic variation on the Y substantially reduces heritable variation for fitness among males, and on a macroevolutionary scale, the Y produces strong selection for genomic rearrangements that move interacting genes onto the nonrecombining region of the Y.

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Dissection of a quantitative trait locus for genetic hypertension on rat chromosome 10.

We have previously identified a locus on rat chromosome 10 as carrying a major hypertension gene, BP/SP-1. The 100:1 odds support interval for this gene extended over a 35-centimorgan (cM) region of the chromosome that included the angiotensin I-converting enzyme (ACE) locus as demonstrated in a cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and the normotensive Wistar-Kyoto (WKY-0HD) rat. Here we report on the further characterization of BP/SP-1, using a congenic strain, WKY-1HD. WKY-1HD animals carry a 6-cM chromosomal fragment genotypically identical with SHRSPHD on chromosome 10, 26 cM away from the ACE locus. Higher blood pressures in the WKY-1HD strain compared with the WKY-0HD strain, as well as absence of linkage of the chromosome 10 region to blood pressure in an F2 (WKY-1HD x SHRSPHD) population suggested the existence of a quantitative trait locus, termed BP/SP-1a, that lies within the SHRSP-congenic region in WKY-1HD. Linkage analysis in the F2 (WKY-0HD x SHRSPHD) cross revealed that BP/SP-1a is linked to basal blood pressure, whereas a second locus on chromosome 10, termed BP/SP-1b, that maps closer to the ACE locus cosegregates predominantly with blood pressure after exposure to excess dietary NaCl. Thus, we hypothesize that the previously reported effect of BP/SP-1 represents a composite phenotype that can be dissected into at least two specific components on the basis of linkage data and congenic experimentation. One of the loci identified, BP/SP-1a, represents the most precisely mapped locus affecting blood pressure that has so far been characterized by random-marker genome screening.

A genome scan for eye color in 502 twin families: Most variation is due to a QTL on chromosome 15q

We have rated eye color on a 3-point scale (1=blue/grey, 2=hazel/green, 3=brown) in 502 twin families and carried out a 5-10 cM genome scan (400-757 markers). We analyzed eye color as a threshold trait and performed multipoint sib pair linkage analysis using variance components analysis in Mx. A lod of 19.2 was found at the marker D15S1002, less than 1 cM from OCA2, which has been previously implicated in eye color variation. We estimate that 74% of variance in eye color liability is due to this QTL and a further 18% due to polygenic effects. However, a large shoulder on this peak suggests that other loci affecting eye color may be telomeric of OCA2 and inflating the QTL estimate. No other peaks reached genome-wide significance, although lods >2 were seen on 5p and 14q and lods >1 were additionally seen on chromosomes 2, 3, 6, 7, 8, 9, 17 and 18. Most of these secondary peaks were reduced or eliminated when we repeated the scan as a two locus analysis with the 15q linkage included, although this does not necessarily exclude them as false positives. We also estimated the interaction between the 15q QTL and the other marker locus but there was only minor evidence for additive x additive epistasis. Elaborating the analysis to the full two-locus model including non-additive main effects and interactions did not strengthen the evidence for epistasis. We conclude that most variation in eye color in Europeans is due to polymorphism in OCA2 but that there may be modifiers at several other loci.

Genetic-epidemiologic analysis of X-inactivation skewing in human females : suggestive evidence for two distinct traits

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Genetic-epidemiologic analysis of X-inactivation skewing in human females : suggestive evidence for two distinct traits

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Cytogenetic characterization of the Passiflora edulis Sims x Passiflora cincinnata Mast. interspecific hybrid and its parents.

The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis x P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/40-60-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of S rDNA sites, and two pairs of 45S rDNA sites that colocalized with two pairs of CMA3 /DAPI- bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.

Ethnic differences in the BMI-%BF relationships between young Japanese and Australian-Caucasian males living in Australia using dual-energy X-ray absorptiometry

The Body Mass Index (BMI) has been used worldwide as an indicator of fatness. However, the universal cut-off points by the World Health Organisation (WHO) classification may not be appropriate for every ethnic group when consider the relationship with their actual total body fatness(%BF). The application of population-specific classifications to assess BMI may be more relevant to public health. Ethnic differences in the BMI%BF relationship between 45 Japanese and 42 Australian-Caucasian males were assessed using whole body dual-energy X-ray absorptiometry (DXA) scan and anthropometry using a standard protocol. Japanese males had significantly (p<0.05) greater %BF at given BMI values than Australian males. When this is taken into account the newly proposed Asia-Pacific BMI classification of BMI 23 as overweight and 25 as obese may better assess the level of obesity that is associated increased health risks for this population. To clarify the current findings, further studies that compare the relationships across other Japanese populations are recommended.