Parental origin of de novo constitutional deletions of chromosomal band 11p13.

One-half of all cases of Wilms tumor (WT), a childhood kidney tumor, show loss of heterozygosity at chromosomal band 11p13 loci, suggesting that mutation of one allele and subsequent mutation or loss of the homologous allele are important events in the development of these tumors. The previously reported nonrandom loss of maternal alleles in these tumors implied that the primary mutation occurred on the paternally derived chromosome and that it was "unmasked" by loss of the normal maternal allele. This, in turn, suggests that the paternally derived allele is more mutable than the maternal one. To investigate whether germinal mutations are seen with equal frequency in maternally versus paternally inherited chromosomes, we determined the parental origin of the de novo germinal 11p13 deletions in eight children by typing lymphocyte DNA from these children and from their parents for 11p13 RFLPs. In seven of the eight cases, the de novo deletion was of paternal origin. The one case of maternal origin was unremarkable in terms of the size or extent of the 11p13 deletion, and the child did develop WT. Transmission of 11p13 deletions by both maternal and paternal carriers of balanced translocations has been reported, although maternal inheritance predominates. These data, in addition to the general preponderance of paternally derived, de novo mutations at other loci, suggest that the increased frequency of paternal deletions we observed is due to an increased germinal mutation rate in males.     

A novel RING finger-B box-coiled-coil protein, GERP

The RING finger domain occurs in a wide variety of proteins involved in cellular regulation. The polymerase chain reaction was used to search for novel RING finger proteins, using primers derived from expressed sequence tags (ests). A cDNA encoding a novel RING finger protein expressed in brain, lung, breast, placenta, kidney, muscle, and germinal center B cells is described. The human gene is expressed in a variety of tumors, including anaplastic oligodendroglioma and maps to chromosome 10q24.3, a region showing frequent deletion or loss of heterozygosity in glioblastomas. It was therefore designated glioblastoma expressed RING finger protein (GERP). GERP contains an N-terminal RING finger, followed by two B-boxes and a coiled-coil, and thus belongs to the RBCC subfamily of RING finger proteins. The structure of this protein and its mapping to a locus thought to harbor tumor suppressor genes indicates that it may be a new tumor suppressor gene important in gliomas and other malignancies.     

Major histocompatibility complex heterozygosity enhances reproductive success

We investigated how heterozygosity at the major histocompatibility complex (MHC) affects fitness in wild-derived (F2) house mice (Mus musculus musculus). To compare and control for potential confounding effects from close inbreeding and genome-wide heterozygosity, we used mice that were systematically outbred. We assessed how heterozygosity at MHC and background loci (using 15 microsatellite markers on 11 different chromosomes) affects individual survival and reproductive success (RS) in large, semi-natural population enclosures. We found that overall heterozygosity significantly increased RS, and this correlation was entirely explained by heterozygosity at two MHC loci. Moreover, we found that the effects of MHC heterozygosity depend on the level of background heterozygosity, and the benefits of maximal MHC heterozygosity show a curvilinear effect with increasing background heterozygosity. The enhanced RS from MHC heterozygosity was not because of increased survival, and although MHC heterozygosity was correlated with body mass, body mass did not correlate with RS when heterozygosity is controlled. Breeders were more MHC heterozygous than nonbreeders for both sexes, indicating that MHC heterozygosity enhanced fecundity, mating success or both. Our results show that (i) MHC heterozygosity enhances fitness among wild, outbred as well as congenic laboratory mice; (ii) heterozygosity-fitness correlations can potentially be explained by a few loci, such as MHC; (iii) MHC heterozygosity can increase fitness, even without affecting survival, by increasing mating and RS; and (iv) MHC effects depend on background genes, and maximal MHC heterozygosity is most beneficial at intermediate or optimal levels of background heterozygosity.     

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.     

Immune suppression and histophysiology of the immune response

Seven daily intramuscular (im) injections of cortisone acetate (25 mg/Kg b.w.) given to rats or rabbits produced, (i) a pronounced reduction in the numbers of small lymphocytes in thymus-independent areas, (ii) atrophy of the thymic cortex, (iii) atrophy of germinal centres and (iv) a consequent depressed production of germinal centre-derived cells. Lymphocyte depletion was not caused by cell lysis. Moreover cell traffic between peripheral lymphoid organs did not seem to be altered. A revival of the depressed germinal centres in cortisone-treated (inbred) rats could be achieved by a transfer of bone-marrow cell suspensions from normal, cortisone-treated or T-cell-deprived animals. It was concluded that cortisone acetate arrests the migration of B-lymphocytes from the bone marrow to germinal centres in peripheral lymphoid organs, and that the accumulations of lymphoid cells in the bone marrow of cortison-treated animals might be composed of immature or mature T- and B-lymphocytes.     

HETEROZYGOSITY,AGGRESSION, AND POPULATION FLUCTUATIONS IN MEADOW VOLES (MICROTUS PENNSYLVANICUS)

We tested whether variation in heterozygosity could produce cyclic changes in population size in meadow voles (Microtus pennsylvanicus). For this to occur, three conditions must be met: (1) populations are more outbred (heterozygotic) at high than low population density, (2) heterozygotic voles are more aggressive than relatively inbred individuals, and (3) heterozygotic voles have lower reproductive fitness, though being superior at defending resources. We found no evidence that heterozygosity varied with population size or that reproductive success varied with heterozygosity. However, the former test was indirect and relatively weak. We directly measured aggression and heterozygosity of individual voles. Aggression was significantly related to heterozygosity: higher heterozygosity correlated with more aggression in males and less aggression in females. The proportion of variance in aggression that could be explained by heterozygosity was small. These results suggest that changes in population size of meadow voles could not be driven by systematic changes in heterozygosity with population size.     

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel,Synbranchus marmoratus Bloch 1795 (Teleostei,Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.     

Age-specific effect of heterozygosity on survival in alpine marmots, Marmota marmota

The fitness consequences of heterozygosity and the mechanisms underpinning them are still highly controversial. Using capture–mark–recapture models, we investigated the effects of individual heterozygosity, measured at 16 microsatellite markers, on age-dependent survival and access to dominance in a socially monogamous mammalian species, the alpine marmot. We found a positive correlation between standardized multilocus heterozygosity and juvenile survival. However, there was no correlation between standardized multilocus heterozygosity and either survival of older individuals or access to dominance. The disappearance of a significant heterozygosity fitness correlation when individuals older than juveniles are considered is consistent with the prediction that differences in survival among individuals are maximal early in life. The lack of a correlation between heterozygosity and access to dominance may be a consequence of few homozygous individuals attaining the age at which they might reach dominance. Two hypotheses have been proposed to explain heterozygosity-fitness correlations: genome-wide effects reflected by all markers or local effects of specific markers linked to genes that determine fitness. In accordance with genome-wide effects of heterozygosity, we found significant correlations between heterozygosities calculated across single locus or across two sets of eight loci. Thus, the genome-wide heterozygosity effect seems to explain the observed heterozygosity-fitness correlation in the alpine marmot.     

Germinal centers and the B-cell system

Summary Germinal centers of the rabbit appendix were studied for the presence of complement receptors, immunoglobulin and alkaline phosphatase. In popliteal lymph nodes, de novo-developing germinal centers were studied with respect to these markers up to 21 days after sheep red blood cell (SRBC)-stimulation. In addition, the possible presence of antigen (SRBC) receptor-bearing cells in these germinal centers was investigated.The results may be summarized as follows: 1) Germinal centers in the appendix as well as those in popliteal lymph nodes were rich in complement receptor-bearing cells. Complement-receptor density did not significantly change during a germinal-center reaction. 2) Immunoglobulins were present only at very low densities on the surface of lymphoid cells in the densely populated area of germinal centers. In germinal centers of popliteal lymph nodes lymphoid cells in the thinly populated area again showed higher densities. Immunoglobulins possibly complexed with antigen on the surface of follicular dendritic cells were not observed in the initial phase of a germinal center reaction. In contrast, in germinal centers of the appendix, immunoglobulin was present in excessive amounts throughout the thinly populated area, possibly complexed with antigen, which is also abundantly present. 3) Reticular staining patterns of alkaline phosphatase were present in the densely populated area, but absent in the thinly populated area of germinal centers in both appendix and popliteal lymph nodes. Primary follicles and young germinal centers were alkaline phosphatase-negative. 4) Antigen receptor-bearing cells were detected in germinal centers of popliteal lymph nodes as early as 5 days after SRBC-stimulation, reaching a maximum at day 10.In conclusion, with the present experimental approach, microenvironmental differences were shown between the densely populated area and the thinly populated area of germinal centers. However, no indication was obtained for a postulated maturation event of the lymphocytes within germinal centers, or for functional differences that may exist between germinal centers in the appendix and popliteal lymph nodes.     

Ultrastructural changes associated with UV irradiation in the "germinal plasm" of Xenopus laevis

To detect structural changes following UV irradiation in the “germinal plasm,” ultrastructure of the “germinal plasm” was studied in normal and UV-irradiated eggs of Xenopus laevis at the following stages: prior to fertilization, early 2-cell, 32-cell, and late blastula. It was revealed that ultrastructural features of the “germinal plasm” were essentially common between Xenopus laevis and Rana pipiens. That is, the “germinal plasm” is composed primarily of a large aggregation of mitochondria and distinctive electron dense bodies (germinal granules). Irregularly shaped cylinderlike granules (giant germinal granules), having the same internal characteristics as the germinal granules, were found in the “germinal plasm” of all eggs examined.Comparison between normal and UV-irradiated eggs has demonstrated that UV irradiation causes swelling and vacuolation of mitochondria and fragmentation of germinal granules. The suggestion is that the integrity of certain UV-sensitive factor(s), which is involved in maintaining normal structure of germinal granules, is indispensable for the determination of the primordial germ cells.     

Heterosis at Allozyme Loci under Inbreeding and Crossbreeding in PINUS ATTENUATA

The dependence of heterosis at isozyme loci on inbreeding and crossbreeding was studied in 10-yr-old trees of knobcone pine (Pinus attenuata Lemm.). Heterozygosity was determined at 24 polymorphic isozyme loci and related to the rate of vegetative growth and cone production. The inbreds, created by selfpollination, had 46% of the heterozygosity of their mothers; the crossbreds, created by interpopulation crossing, had 155% of the heterozygosity of their mothers. Within the crossbreds, heterozygosity was positively correlated with trunk growth, but negatively correlated with cone production. Results in the crossbreds, however, were strongly influenced by a few individuals that showed unusually slow growth, high reproduction and low heterozygosity. Without those individuals, there was no relationship of heterozygosity to either growth or reproduction.—Within the inbreds, heterozygosity was positively correlated with both trunk growth and cone production. Each locus that was heterozygous in the mothers was calculated to mark about 3% of the genome for identity by descent in the inbred progeny; the total proportion of the genome marked was between 10 and 11%. Using these estimates to relate heterozygosity to the inbreeding coefficient (F) gave estimates of inbreeding depression per unit of F that fell within the range of published values for conifers. The strength of heterosis found among the inbreds suggests that single-locus or multilocus overdominance should be exceedingly difficult to detect in natural populations of predominantly outcrossing species.     

Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes.

In this study, the effect of different energy sources used in Eagle's minimum essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5.5 mmol 1(-1)), pyruvate (0.23 mmol 1(-1)) and glutamine (2 mmol 1(-1)) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 mumol dibutyryl cAMP 1(-1) during 17-18 h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99-100% germinal vesicle breakdown); all other combinations resulted in < or = 54% germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (< or = 10% GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (> or = 86% viability in pyruvate-containing medium; < or = 35% viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (> or = 89% germinal vesicle breakdown) and viability (> or = 91%). The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5-33% germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon D-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon D-glucose. Uptake and metabolism of D-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of D-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of D-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) D-mannose, a glycolysable sugar, was as effective as D-glucose in supporting the ligand effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Angiogenic inhibition reduces germinal matrix hemorrhage

The germinal matrix of premature infants is selectively vulnerable to hemorrhage within the first 48 h of life. To assess the role of vascular immaturity in germinal matrix hemorrhage (GMH), we evaluated germinal matrix angiogenesis in human fetuses and premature infants, as well as in premature rabbit pups, and noted active vessel remodeling in all three. Vascular endothelial growth factor (VEGF), angiopoietin-2 and endothelial cell proliferation were present at consistently higher levels in the germinal matrix relative to the white matter anlagen and cortical mantle. On that basis, we asked whether prenatal treatment with either of two angiogenic inhibitors, the COX-2 inhibitor celecoxib, or the VEGFR2 inhibitor ZD6474, could suppress the incidence of GMH in premature rabbit pups. Celecoxib treatment decreased angiopoietin-2 and VEGF levels as well as germinal matrix endothelial proliferation. Furthermore, treatment with celecoxib or ZD6474 substantially decreased the incidence of GMH. Thus, by suppressing germinal matrix angiogenesis, prenatal celecoxib or ZD6474 treatment may be able to reduce both the incidence and severity of GMH in susceptible premature infants.     

Germinal centers and the B-cell system

Summary The migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T-and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with ³H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.     

Genome-scale patterns in the loss of heterozygosity incidence in Saccharomyces cerevisiae

Former studies have established that loss of heterozygosity can be a key driver of sequence evolution in unicellular eukaryotes and tissues of metazoans. However, little is known about whether the distribution of loss of heterozygosity events is largely random or forms discernible patterns across genomes. To initiate our experiments, we introduced selectable markers to both arms of all chromosomes of the budding yeast. Subsequent extensive assays, repeated over several genetic backgrounds and environments, provided a wealth of information on the genetic and environmental determinants of loss of heterozygosity. Three findings stand out. First, the number of loss of heterozygosity events per unit time was more than 25 times higher for growing than starving cells. Second, loss of heterozygosity was most frequent when regions of homology around a recombination site were identical, about a half-% sequence divergence was sufficient to reduce its incidence. Finally, the density of loss of heterozygosity events was highly dependent on the genome’s physical architecture. It was several-fold higher on short chromosomal arms than on long ones. Comparably large differences were seen within a single arm where regions close to a centromere were visibly less affected than regions close, though usually not strictly adjacent, to a telomere. We suggest that the observed uneven distribution of loss of heterozygosity events could have been caused not only by an uneven density of initial DNA damages. Location-depended differences in the mode of DNA repair, or its effect on fitness, were likely to operate as well.     

Statistical Studies on Protein Polymorphism in Natural Populations I. Distribution of Single Locus Heterozygosity

Surveying the literature, the frequency distribution of single-locus heterozygosity among protein loci was examined in 95 vertebrate and 34 invertebrate species with the aim of testing the validity of the mutation-drift hypothesis. This distribution did not differ significantly from that expected under the mutation-drift hypothesis for any of the species examined when tested by the Kolmogorov-Smirnov goodness-of-fit statistic. The agreement between the observed interlocus variance of heterozygosity and its theoretical expectation was also satisfactory. There was an indication that variation in the mutation rate among loci inflates the interlocus variance of heterozygosity. The variance of heterozygosity for a homologous locus among different species was also studied. This variance generally agreed with the theoretical value very well, though in some groups of Drosophila species there was a significant discrepancy. The observed relationship between average heterozygosity and the proportion of polymorphic loci was in good agreement with the theoretical relationship. It was concluded that, with respect to the pattern of distribution of heterozygosity, the majority of data on protein polymorphisms are consistent with the mutation-drift hypothesis. After examining alternative possible explanations involving selection, it was concluded that the present data cannot be explained adequately without considering a large effect of random genetic drift, whether there is selection or not.     

Studies on the germinal center

Summary Ultrastructure of mitotic cells in human lymph node germinal centers was deliberately studied in contrast to that of plasma cells in mitosis which were rarely found in medullary cords or lymphatic sinuses of the same materials. It was demonstrated that the mitotic cells in germinal centers are evidently different from the latter in the absence of lamellary arranged endoplasmic reticulum with ribosomes and Golgi apparatus, and are quite similar to the ultrastructure of thymic lymphocytes in mitosis reported by Murray et al. It should be concluded from these findings that the cells produced locally within the germinal centers in human lymph nodes are lymphocytic as has been repeatedly suggested by the authors.Supported by Grant in Aid from the Ministry of Education of Japan (69-9254).     

Distribution of the Germinal Vesicle Material during Progesterone-Induced Oocyte Maturation in Xenopus and in Cynops

The distribution of the germinal vesicle material in the oocyte during progesterone-induced maturation was studied in Xenopus and in Cynops. In both species, two distinctive masses of yolkfree cytoplasm appear in specific areas of the oocyte and at definite stages of maturation. One, the primary cytoplasmic mass, is formed at the basal side of the germinal vesicle during early maturation and is very RNA-rich. In Xenopus , a large part of the primary cytoplasmic mass persists as a mass during maturation and ends up as a thin disk at the boundary between the animal and the vegetal hemisphere in the mature oocyte. In Cynops , a rod-like primary cytoplasmic mass extends near to the equatorial zone and becomes indistinct in the mature oocyte. The other, the secondary cytoplasmic mass, is formed at or prior to germinal vesicle breakdown in areas around the germinal vesicle and is also RNA-rich. The secondary cytoplasmic mass is dispersed and constitutes the RNA-rich animal hemisphere cytoplasm in the mature oocyte. Observed results suggest that the primary and the secondary cytoplasmic mass contain different germinal vesicle materials.