4th Chromosome Research Articles

The MSL complex levels are critical for its correct targeting to the chromosomes in Drosophila melanogaster

In Drosophila, dosage compensation requires assembly of the Male Specific Lethal (MSL) protein complex for doubling transcription of most X-linked genes in males. The recognition of the X chromosome by the MSL complex has been suggested to include initial assembly at approximately 35 chromatin entry sites and subsequent spreading of mature complexes in cis to numerous additional sites along the chromosome. To understand this process further we examined MSL patterns in a range of wild-type and mutant backgrounds producing different amounts of MSL components. Our data support a model in which MSL complex binding to the X is directed by a hierarchy of target sites that display different affinities for the MSL proteins. Chromatin entry sites differ in their ability to provide local intensive binding of complexes to adjacent regions, and need high MSL complex titers to achieve this. We also mapped a set of definite autosomal regions (approximately 70) competent to associate with the functional MSL complex in wild-type males. Overexpression of both MSL1 and MSL2 stabilizes this binding and results in inappropriate MSL binding to the chromocenter and the 4th chromosome. Thus, wild-type MSL complex titers are critical for correct targeting to the X chromosome.

DNA profiling by detection of repetitive nucleotide sequences on human chromosome 6.

Genetic/genomic polymorphism, i.e. variations in DNA sequences are ideally assayed by direct nucleotide sequencing of a gene region or other homologous segment of the genome. An easier and cheaper approach, however, if the variants are analyzed by hybridization technology using restriction fragment length polymorphisms (RFLPs) or by detection of the number of tandem repeats (VNTR) of small DNA segments, the "minisatellites". In this study we describe results of the DNA analysis of repetitive sequences of human 6th chromosome by the application of a chemiluminescent labeled probes. The allele frequency distribution of polymorphic DNA sequences has been determined in unrelated individuals. The isolated genomic DNA was cut with Pst I restriction enzyme, size fractionated on agarose gel and hybridized with a chemiluminescent labeled D6 S132 probe. At this locus the Pst I cleaved DNA fragments are ranging from 1841 to 6098 base pairs (bp). Specific genetic pattern was characterized by more frequent fragments (3313 and 3884 bp), and the rarely occurring ones (clustered between 1841-2595 and 5227-6098 bp). Our study provides a further possibility for characterization of individual genomic patterns.

GIP, a G-protein-coupled receptor interacting protein.

A novel protein was cloned while screening for partners interacting with the second intracellular loop of the V2 vasopressin receptor (V2R). The protein was named GIP as in G-protein-coupled receptor Interacting Protein; the corresponding gene was located on the 17th chromosome where three exons encode for a 379-amino-acid protein.GIP subcellular localization was studied by immunocytochemistry and also using a biotinylating agent. The protein was found to be localized, at least in part, on the plasma membrane, probably in the form of a trimer. The results indicated that GIP is a transmembrane protein and the most part of the molecule is intracellular. Sequence homology inferred that GIP cytosolic domain is folded as a collagen-like helix followed by a globular domain. The interaction of the globular domain with the V2R was confirmed by pull-down experiments indicating that this structural motif can also interact with cytosolic proteins.

Chromosomal and allelic variation in Drosophila americana: selective maintenance of a chromosomal cline.

Geographically structured genetic variation, as represented by clines and hybrid zones, offers unique opportunities to study adaptation and speciation in natural populations. A hybrid zone has been reported between Drosophila americana americana and Drosophila americana texana, two taxa that are distinguished solely by the arrangement of their X and 4th chromosomes. In this study, samples of D. americana were collected along a latitudinal transect across the inferred hybrid zone, and the frequency of the alternative chromosomal arrangements is reported. These data illustrate that the alternative chromosomal arrangements are distributed along a shallow cline over a broad geographic region, and that the frequency of the arrangements is tightly correlated with latitude. Allelic variants at 13 RFLP loci in three genes on chromosome 4 exhibit no evidence of association with the cline. Presence of a cline for the chromosomal arrangements, as well as a general absence of geographic structure for variation at these genes, is interpreted as evidence that natural selection is responsible for the maintenance of this chromosomal cline. Furthermore, these results demonstrate that taxonomic subdivision of D. americana is unwarranted, because it exists as a cohesive species that is segregating a chromosomal fusion.

Frequency and distribution of t-haplotypes in the Southeast Asian house mouse (Mus musculus castaneus) in Taiwan.

t-haplotypes are a meiotic drive system found on the 17th chromosome of the house mouse (Mus musculus). They can be found in wild populations of all four genetically differentiated subspecies. The drive phenomenon is male-specific, such that heterozygous males (+/t) show non-Mendelian transmission and transmit the t-chromosome to > 90% of their offspring. So far the most comprehensive studies on the frequencies of t-haplotypes in natural populations have been on just one of the subspecies (M. musculus domesticus). We applied molecular methods to accurately screen t-haplotypes in a large number of populations of a second subspecies (M. musculus castaneus) distributed in Taiwan. We found that the overall t-haplotype frequency is low in M. m. castaneus (0.108), and the chromosomes are patchily distributed among its populations, closely resembling the situation found in M. m. domesticus. Further, we found the frequencies of t-haplotypes in our sample did not differ in relation to the sex or age of mice. This resemblance in the frequency and distribution among populations of the two distinct subspecies suggests that similar general mechanisms might be responsible for the low frequencies in both subspecies.

Karyological study on four endemic Ebenus L. taxa (Leguminosae) in Turkey

Abstract This work examines the karyology of four taxa of Ebenus L.: E. plumosa Boiss. & Bal. var. plumosa, E. plumosa var. speciosa Hub.-Mor., E. macrophylla Jaub. & Spach and E. barbigera Boiss. All species are diploid with 2n=14 chromosomes. Chromosome lengths in these four Ebenus species are between 4.30 and 2.67 μm, 4.91 and 3.09 μm, 4.22 and 2.15 μm, 2.78 and 1.58 μm, respectively. The karyotype of E. plumosa var. plumosa consists of 1m+1sm+5m, of E. plumosa var. speciosa consists of 2m+1sm+4m. The karyotypes of the other two species consist of 7m. Satellites are on the long arms of Chromosome 4 in E. plumosa var. plumosa and E. macrophylla and on the short arms in E. plumosa var. speciosa. In E. barbigera satellites are on the long arms of Chromosome 3. There is a 2.44 μm long chromosome fragment on the short arm on one of the homologous 6th Chromosome in E. plumosa var. speciosa.

Isolation and mapping of bacterial artificial chromosome (BAC) clone containing telomere-associated sequence

Using single primer (TTTAGGG)(3) or (CCCTAAA)(3)CCC corresponding to rice telomeric repeat sequences, two fragments were amplified from rice genomic DNA and named Tas1 and Tas2. These two fragments did not show polymorphism between the mapping parents. Tasl was found to hybridize to rice telomeric regions by in situ hybridization (FISH). Then a rice bacterial artificial chromosome (BAC) library was constructed and Tasl was used to screen it. One positive clone was identified. A single copy sequence of this BAC clone was mapped at the end of the 6th chromosome of rice.

Unaltered Meiotic Chromosome Segregation in Drosophila melanogaster Raised on a 5% Quercetin Diet

Flavonoid plant pigments are an integral part of the human diet. Although potentially negative mitotic effects of flavonoids have been observed in model organisms, investigation into meiotic effects of flavonoids has been neglected. As flavonoids affect cell signalling and DNA replication, and because the flavonoid content of the human food supply is being increased, determining the effects of flavonoids on meiotic fidelity is important. Here, the effect of the human food supply's most prevalent flavonoid, quercetin, on the level of meiotic recombination and the amount of X and 4th chromosome non-disjunction in Drosophila melanogaster females was determined. This model organism was chosen since Drosophila melanogaster and Homo sapiens share a remarkable number of commonalties in the meiotic processes of oogenesis and because genetic techniques allow a detailed analysis of meiotic processes in Drosophila. No significant effect on either non-disjunction levels or the percentage distribution of exchange bivalents was observed. A significant effect was observed on the number of offspring; F 1 and F 2 generations of flies raised on a quercetin diet produced over 10% more progeny than flies raised on a control diet. In this investigation, high quercetin consumption by Drosophila melanogaster females did not pose a threat to meiotic fidelity.

Study on the relationship between tumour necrosis factor gene polymorphism and prognosis in the patients with renal cell carcinoma

Clinical significance of polymorphism of tumour necrosis factor (TNF) genes encoded on the short arm of the 6th chromosome in the patients with renal cell carcinoma (RCC) has not been evaluated well so far. We studied on the TNF genes polymorphism of RCC focusing on the relationship between the genetic polymorphism and the prognosis. The subjects were seventy-three patients with RCC treated at our hospitals during the past 20 years. The genomic DNA was examined by the methods of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from frozen peripheral blood of these patients. The items examined were the genetic polymorphisms of TNF-alpha (alpha 1, alpha 2) and TNF-beta (beta 1, beta 2), and we tried to study on the prognostic outcome of RCC based upon each zygote of TNF. 1) The proportion of TNF-alpha and TNF-beta polymorphisms: We observed TNF-alpha 1/1 homozygote in 71 patients (97.3%). As to TNF-beta polymorphism, we observed TNF-beta 2/2 homozygote in 33 patients (45.2%), TNF-beta 1/2 heterozygote in 31 (42.5%) and TNF-beta 1/1 homozygote in 9 (12.3%). The proportion of TNF-beta polymorphism was almost the same as that of healthy Japanese. 2) PROGNOSIS: Regarding the 17-year survival, all patients with TNF-beta 1/1 homozygote were alive, and we observed a significantly favourable prognosis in the patients with TNF-beta 1/1 homozygote compared with other zygotes of TNF-beta polymorphism. The reasons for these favourable prognosis were thought that the patients with TNF-beta 1/1 homozygote showed much lower stage and/or grade than those of other zygotes. We conclude that the TNF-beta gene polymorphism is a useful marker for understanding the prognosis of RCC and a part of cellular immunity related to the tumour and its host.

ADrosophilaKinesin-like Protein, Klp38B, Functions during Meiosis, Mitosis, and Segmentation

We show thatklp38B,isolated as a mutation that dominantly prolongs blastoderm mitotic cycles in Drosophila, encodes a Drosophila kinesin-like protein. Further genetic analyses show that Klp38B not only functions during mitosis, but is also required for meiosis and abdominal segmentation. Sequence comparisons suggest that Klp38B encodes an amino-terminal microtubule motor domain, a central α-helical coiled-coil domain, and a C-terminal globular domain. Evidence that Klp38B is required during meiosis is that flies transheterozygous for mutations in bothklp38Bandnodhave a high frequency of 4th chromosome meiotic nondisjunction. Nod is a chromokinesin, a chromosome binding kinesin, that is believed to provide astral-exclusion forces during the metaphase stage of meiosis. Evidence that Klp38B is required during mitosis is that embryos from female germline clones ofklp38Bmutations have holes in the cuticle similar to a zygoticstring(dCDC25) phenotype. Also, anti-Klp38B antibody injection into precellularization blastoderm embryos causes developmental arrest and the formation of circular mitotic figures. We speculate, based on these phenotypes, that Klp38B is a chromokinesin that provides astral-exclusion forces on the chromosomes during meiosis and mitosis. Consistent with this hypothesis, we have identified an HMG-1 homologous region on Klp38B that could potentially bind AT-rich DNA sequences. Finally, we show thatklp38Bmutations have defects in abdominal segmentation, suggesting that Klp38B, like Xenopus chromokinesin Xklp1, might be involved in polar granule formation.

Integrated Epstein‐Barr virus (EBV) and chromosomal abnormality in chronic active EBV infection

In order to examine the role of Epstein-Barr virus (EBV) in the pathogenesis of chronic active EBV infection (CAEBV), we investigated whether or not EBV integration into the human genome is associated with any chromosonal abnormality. We therefore analyzed 4 cases of CAEBV: 2 cases showed a normal karyotype, while one had an oligo-clonal 6th chromosomal abnormality and the fourth had a clonal 6th deletion (q15q23). In addition, the case with an oligo-clonal abnormality also had oligo-clonal EBV terminal repeat (TR) bands, while the case with a clonal abnormality showed a clonal TR band. In contrast, the 2 cases with a normal karyotype showed no clonal band. Two-color fluorescence in situ hybridization (FISH) was used to detect the integrated EBV and the 6th chromosomal site. The presence of integrated EBV into the 6th chromosome was not frequent in the 2 cases with a normal karyotype, but it was statistically frequent in the case with an oligo-clonal 6th abnormality. In the case with a clonal 6th deletion, integration in the 6th chromosome was also slightly higher than that in the other chromosomes. In CAEBV, integrated EBV might thus be associated both with chromosomal abnormality and with pathogenesis. Int. J. Cancer 71: 943-947, 1997. © 1997 Wiley-Liss Inc.

Integrated Epstein-Barr virus (EBV) and chromosomal abnormality in chronic active EBV infection.

In order to examine the role of Epstein-Barr virus (EBV) in the pathogenesis of chronic active EBV infection (CAEBV), we investigated whether or not EBV integration into the human genome is associated with any chromosonal abnormality. We therefore analyzed 4 cases of CAEBV: 2 cases showed a normal karyotype, while one had an oligo-clonal 6th chromosomal abnormality and the fourth had a clonal 6th deletion (q15q23). In addition, the case with an oligo-clonal abnormality also had oligo-clonal EBV terminal repeat (TR) bands, while the case with a clonal abnormality showed a clonal TR band. In contrast, the 2 cases with a normal karyotype showed no clonal band. Two-color fluorescence in situ hybridization (FISH) was used to detect the integrated EBV and the 6th chromosomal site. The presence of integrated EBV into the 6th chromosome was not frequent in the 2 cases with a normal karyotype, but it was statistically frequent in the case with an oligo-clonal 6th abnormality. In the case with a clonal 6th deletion, integration in the 6th chromosome was also slightly higher than that in the other chromosomes. In CAEBV, integrated EBV might thus be associated both with chromosomal abnormality and with pathogenesis.

Cytogenetic characteristics of a population of Chironomus riparius Meigen 1804 (Diptera, Chironomidae) from a polluted Po river station

A population of Chironomus riparius from a Po river station near Moncalieri (a trace-metal polluted station) was studied. In this population was established a great variability of band structure of polytene chromosomes as well as paracentric heterozygous inversions, deletions, deficiencies, partial breaks, diploid chromosome fragments, and changes in functional activity and appearance of heterochromatin. In arms A through F, some bands had an increased size compared to the standard chromosomic map. Some bands appeared in a heterozygous or normal homozygous state or were amplified. In all arms, many condensed stable bands appeared in the decondensed state when compared to the standard map. Asynaptic zones in arms E and G as well as heterozygous Balbiani rings and NORs were established. Very often the 4th chromosome was almost completely heteropycnotic and looded like a ‘pompon’ chromosome. For the first time in this species, a high frequency of ectopic pairings of different arms was observed. Telomeric regions involved in ectopic pairings had a granular appearance, as did some centromeres. The hypothesis is advanced that such a high frequency of structural rearrangements could be correlated with genomic distribution of specific mobile elements.

Linkage Group Analysis of Pyraclofos Resistance in the Housefly, Musca domestica L.

A strain of housefly, YBOL, highly resistant to pyraclofos ((R, S)-[O-1-(4-chlorophenyl)pyrazol-4-yl O-ethyl S-n-propyl phosphorothioate], Boltage[○!R]), was established from a colony collected from 3rd Yumenoshima after selection with this chemical. The reciprocal crosses between the resistant (YBOL) and susceptible strains showed that pyraclofos resistance was incomplete dominance. Linkage group analysis of the dominant factors responsible for this resistance was carried out by the F1 male backcross method using a susceptible multichromosomal marker strain. The dominant factors for pyraclofos resistance were located on the 2nd, 4th and 5th chromosomes. The factors on the 2nd chromosome contributed the greatest to pyraclofos resistance, followed by those on the 5th chromosome and then the 4th chromosome. In pyraclofos resistant strain, it was revealed that the male determinant factor (M) existed on the 3rd chromosome.

Comparative karyotype analysis of 24-chromosomeRanaspecies (R. chensinensisandR. arvalis, Amphibia, Anura)

SUMMARYResults of a comparative study of the karyotypes of Rana chensinensis and R. arvalis are reported. On the basis of detail morphometrical analysis of each chromosome pair significant karyotype differences were established between two species concerning localization of secondary constriction, the centromere position of 6th chromosome pair and the absolute length of 3 chromosome pairs. The possible mechanisms giving rase to the differences between both karyotypes were discussed. It was suggested that karyotypic divergence of R. arvalis was caused by a pericentric inversion, independent local deletions and transpositions with a consequent amplification of NOR-sites.

Involvement of regions of the 4th and 7th chromosomes in the open-field activity of mice

Reactivity to a new environment was studied in mice, using an open-field procedure in two strains, C57BL/6By and ABP/Le, the F 1 populations and the intercrosses F 2 and backcross segregating populations. The analysis of the behavioral traits: peripheral and central activities, leaning, rearing and defecation in the parental strains made it possible to show that the ABP/Le strain was more reactive than C57BL/6By. In addition, the study of segregating, for four phenotypic markers, in F 2 and backcross populations strongly suggested that two autosomal regions were involved in the control of open-field behavior: one in chromosomal region comprising the b locus on chromosome 4 and one in chromosomal region comprising the p locus on chromosome 7.

Lipopolysaccharide‐stimulated events in B Cell Activation

High concentrations of thymus-independent (TI) antigens are capable of inducing polyclonal B cell activation by their intrinsic mitogenic properties, irrespective of the specificity of the Ig receptors. Due to a genetic defect on the 4th chromosome, B cells from C3H/HeJ mice do not respond to lipopolysaccharide (LPS). In order to define at which step the mutation affects the signalling pathway, we compared B cells from C3H/HeJ and CBA mice with regard to changes of three events, namely cell size, and MHC class I and II antigen expression after LPS stimulation. We found that cell size and expression of MHC antigens increase in B cells from CBA mice after LPS stimulation, whereas B cells from C3H/HeJ mice do not respond at all. This suggests that the defect in C3H/HeJ mice interferes with early events in the signalling pathway, either due to the absence of a LPS receptor on B cell surface or the lack of an initial component necessary for effective signal transmission subsequent to LPS receptor binding. Our results also have shown that stimulation of anti-Ig antibodies and LPS differ in some signalling events and have different final effects on B cells, which suggests that they may function differently via distinct signalling pathways.

Characterization of vincristine-resistant HOB1 lymphoma cell line showing the classical MDR phenotype and altered expression of membrane glycoproteins.

A multidrug-resistant (MDR) cell line isolated from HOB1 lymphoma cells was characterized. The MDR phenotype in this cell line was typified by resistance to vincristine with varying degrees of cross-resistance to Adriamycin, colcemid and actinomycin D. Decreased intracellular [3H]vincristine with concurrent increase in the expression of a 170-kDa membrane glycoprotein (P-glycoprotein) suggested a plausible underlying mechanism for the development of resistance. Amplification of the mdr1 gene as well as a homogeneous staining region on the long arm of the 7th chromosome was observed. Moreover, metabolic studies with [14C]glucosamine or [14C]mannose indicated differential expressions of membrane glycoproteins between the drug-sensitive parental and drug-resistant descendant cells. It is concluded that the development of drug resistance in HOB1 lymphoma cells was strongly correlated with the overexpression of P-glycoprotein.

There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology.

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.

Meiosis in pollen mother cells of 15 Cryptomeria japonica clones with special emphasis on the behavior of the 6th chromosome pair heteromorphic to chromomycin A3 band.

Meioses in pollen mother cells (PMCs) were examined by the conventional staining and the CMA-staining methods in 15 clones of Cryptomeria japonica D. Don carrying three different karyotypes identified and classified by the characteristics of interstitial chromomycin A3 (CMA)-band or secondary constriction on the 6th chromosome pair. Before fixation of PMCs, a newly developed hypotonic treatment was applied for easy and constant preparation of well-spread meiotic chromosomes. At meiotic metaphase I 11 bivalents were observed, which possessed chiasmata at the interstitial region and/or the distal end. Although the CMA-bands were clearly observed on meiotic chromosomes, no chiasma was formed involving these bands. Most of the clones exhibited normal meiotic progression, but two clones (Syusou 5 and 11) showed some abnormal meioses. In Syusou 5 the heteromorphic 6th chromosome pair was involved in univalent formation more frequently than the other chromosome pairs. The normal meiosis in most clones with the heteromorphic 6th chromosome pair indicates that the heteromorphic chromosomes do not affect meiotic process.