Allele Deletion Mapping of the Short Arm of Human Chromosome 3 in Renal Carcinoma

Allelic deletions along the short arm of human chromosome 3 were mapped in 57 pairs of DNA samples from tumor and normal tissue of renal carcinoma patients in order to locate potential tumor suppressor genes. Twenty highly polymorphic microsatellite markers were used for deletion mapping. Allelic deletions were found in most of the samples (91%). Extended terminal deletions (56%) prevailed over shorter internal and multiple deletions and dominated (65%) in the most aggressive histopathological kidney cancer subtype, clear-cell carcinoma. Frequency analysis of loss of heterozygosity allowed detection of the human chromosome 3 regions most essential for renal carcinomas: the region adjacent to the gene VHL(3p26–p25), the region of homozygous deletions AP20 (3p22–p21.33), and a new region between markers D3S2420 and D3S2409 (3p21.31, 2.2 Mbp).     

Ring 2 chromosome associated with failure to thrive,microcephaly and dysmorphic facial features

We report here a child with a ring chromosome 2 [r(2)] associated with failure to thrive, microcephaly and dysmorphic features. The chromosomal aberration was defined by chromosome microarray analysis, revealing two small deletions of 2p25.3 (139 kb) and 2q37.3 (147 kb).     

Increased FGF3 and FGF4 gene dosage is a risk factor for craniosynostosis

Interstitial duplications involving chromosome 11q have rarely been reported in the literature and mainly represent large, cytogenetically detectable rearrangements associated with a wide and variable spectrum of neurodevelopmental disorders. We report on a patient affected by intellectual disability, craniosynostosis, and microcephaly. Array-CGH analysis identified a de novo 290 kb interstitial duplication of chromosome 11q13.3 including the FGF3 and FGF4 genes. Clinical comparison of our patient with those previously reported with overlapping 11q duplications allows us to define the minimal duplicated region associated with craniosynostosis and strongly supports the hypothesis that the constitutional increased dosage of the FGF3 and FGF4 genes is a risk factor for craniosynostosis in humans.     

Chromosome‐specific microsatellites from the tammar wallaby X chromosome and chromosome 2

Microsatellites were identified from fully sequenced bacterial artificial chromosome clones from the X chromosome and chromosome 2 of the tammar wallaby, Macropus eugenii. A total of 20 microsatellites were genotyped. Seven X chromosome loci and eight loci from chromosome 2 were polymorphic, with between two and 11 alleles per locus. These markers will facilitate genetic mapping in the tammar wallaby and will have wider applications in macropod population genetics. In particular, X chromosome microsatellites will provide a novel evolutionary perspective from which to view macropod mating systems and gene flow.     

PIF1 helicase promotes break‐induced replication in mammalian cells

Break‐induced replication (BIR) is a specialized homologous‐recombination pathway for DNA double‐strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP‐based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1‐dependent BIR is used for homology‐initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation‐prone AT‐rich DNA sequences derived from common fragile sites (CFS‐ATs) induce BIR upon replication stress and oncogenic stress, and PCNA‐dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC‐mediated repair of double‐ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease‐generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS‐ATs. The breast cancer‐associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.     

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.     

Efficient Amplification of Insert End Sequences from Bacterial Artificial Chromosome Clones by Thermal Asymmetric Interlaced PCR

TAIL-PCR is a powerful tool for the recovery of DNA fragments adjacent to known sequences. A protocol is presented for the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. Abbreviations: AD primer, arbitrary degenerate primer; BAC, bacterial artificial chromosome.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Effect of Temperature on Fecundity,Life Span and Morphology of Long‐ and Short‐Spined Clones of Brachionus caudatus f. apsteini (Rotifera)

We investigated the effect of temperature (20, 25 and 30 °C) on fecundity, life span and morphology of the rotifer Brachionus caudatus f. apsteini. For each temperature, short posterior‐spined and long posterior‐spined clones of B. caudatus f. apsteini were individually cultured for up to six generations. The rotifers were fed Chlorella sp. at a density of 1 × 10⁶ cells ml^–1. Morphometric data (body size and spine length) were collected. Total number of offspring producing by a single female per life cycle at high temperature was higher than at low temperature. The duration of juvenile period, reproductive period, post‐reproductive period and life span of both clones of B. caudatus f. apsteini decreased with increasing temperature. All offspring of short posterior‐spined clone produce posterior spines at 20 and 25 °C, with an average length of 19.8 ± 6.6 and 11.9 ± 2.6 μm, respectively. In contrast, they cannot develop posterior spines at 30 °C, at which the average length of the posterior spine remnant was 6.4 ± 1.3 μm. On the other hand, all offspring of long posterior‐spined clone have long posterior spines with average lengths of 36.8 ± 6.1, 36.3 ± 5.2 and 36.6 ± 6.2 μm at 20, 25 and 30 °C, respectively. This study indicated that the production of posterior spines can be induced by low temperature and that short posterior‐spined and long posterior‐spined clones are genetically different. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular evidence that the genes for dioecism and monoecism in Spinacia oleracea L. are located at different loci in a chromosomal region

Spinach (Spinacia oleracea L.) is widely known to be dioecious. However, monoecious plants can also occur in this species. Sex expression in dioecious spinach plants is controlled by a single gene pair termed X and Y. Our previous study showed that a single, incompletely dominant gene, which controls the monoecious condition in spinach line 03–336, should be allelic or linked to X/Y. Here, we developed 19 AFLP markers closely linked to the monoecious gene. The AFLP markers were mapped to a 38.2-cM chromosomal region that included the monoecious gene, which is bracketed between flanking markers with a distance of 7.1 cM. The four AFLP markers developed in our studies were converted into sequence-characterized amplified region (SCAR) markers, which are linked to both the monoecious gene and Y and are common to both populations segregating for the genes. Linkage analysis using the SCAR markers suggested that the monoecious gene (M) and Y are located in different intervals, between different marker pairs. Analysis of populations segregating for both M and Y also directly demonstrates linkage of the genes at a distance of ∼12 cM. The data presented in this study may be useful for breeding dioecious and highly male monoecious lines utilized as the pollen parents for hybrid seed production, as well as for studies of the evolutionary history of sexual systems in this species, and can provide a molecular basis for positional cloning of the sex-determining genes.     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

红羊新蚤的地理分布、宿主关系和鉴别特征的研究(蚤目：多毛蚤科)

自从红羊新蚤Neopsylla hopgyangensis Li,Bai & chen,1986从宁夏回族自治区的海原和贺兰山林区发现并报道(四川动物5(2)：1-3)以后,青海、甘肃和陕西均先后发现他们以往鉴定为二齿新蚤的大多数为红羊新蚤。本文报道红羊新蚤的地理分布、海拔、主要宿主及鉴别特征的应用。     

The obesity in bilateral ovariectomized rats is related to a decrease in the expression of leptin receptors in the brain.

We investigated the expression levels of leptin receptors in the brain of ovariectomized (OVX) rats. The mean expression level of ob mRNA in adipose tissues of OVX rats was significantly (P < 0.01) lower than that in the SHAM operation group rats, and the mean body weight of OVX rats was significantly (P < 0.01) greater than that in the SHAM group rats. However, there were no differences between serum leptin concentrations in these two groups. The mean level of leptin receptor (OB-R) mRNA expression in the brain tissue and the mean level of long form type OB-R (OB-RL) mRNA expression in the hypothalamus of the OVX rats were significantly (P < 0.05) lower than those in the SHAM group rats. These changes were cancelled by supplementation with 17 beta-estradiol in OVX rats. These results suggested that not only changes in the expression level of ob mRNA in adipose tissue and the serum leptin concentration but also changes in the OB-R mRNA in the brain are involved in the body weight increase in OVX rats and that a decrease in OB-R makes transmission of signals to suppress the amount of food intake difficult, thus leading to an increase in body weight.     

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird‐of‐paradise

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat‐rich and GC‐rich regions (genomic “dark matter”) limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long‐read, linked‐read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC‐rich microchromosomes and the repeat‐rich W chromosome. Telomere‐to‐telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.     

Mapping breakpoints of a familial chromosome insertion (18,7) (q22.1; q36.2q21.11) to DPP6 and CACNA2D1 genes in an azoospermic male

It is widely accepted that the incidence of chromosomal aberration is 10–15.2% in the azoospermic male; however, the exact genetic damages are currently unknown for more than 40% of azoospermia. To elucidate the causative gene defects, we used the next generation sequencing (NGS) to map the breakpoints of a chromosome insertion from an azoospermic male who carries a balanced, maternally inherited karyotype 46, XY, inv ins (18,7) (q22.1; q36.2q21.11). The analysis revealed that the breakage in chromosome 7 disrupts two genes, dipeptidyl aminopeptidase-like protein 6 (DPP6) and contactin-associated protein-like 2 (CACNA2D1), the former participates in regulation of voltage-gated potassium channels, and the latter is one of the components in voltage-gated calcium channels. The deletion and duplication were not identified equal or beyond 100 kb, but 4 homologous DNA elements were verified proximal to the breakpoints. One of the proband's sisters inherited the same aberrant karyotype and experienced recurrent miscarriages and consecutive fetus death, while in contrast, another sister with a normal karyotype experienced normal labor and gave birth to healthy babies. The insertional translocation is confirmed with FISH and the Y-chromosome microdeletions were excluded by genetic testing. This is the first report describing chromosome insertion inv ins (18,7) and attributes DPP6 and CACNA2D1 to azoospermia.     

The origin of a selfish B chromosome triggering paternal sex ratio in the parasitoid wasp Trichogramma kaykai

This study uses molecular and cytogenetic methods to determine the origin of a B chromosome in some males of the wasp Trichogramma kaykai. This so-called paternal sex ratio (PSR) chromosome transmits only through sperm and shortly after fertilization triggers degeneration of the paternal genome, while keeping itself intact. The resulting embryos develop into haploid B-chromosome-carrying males. Another PSR chromosome with a very similar mode of action is found in the distantly related wasp Nasonia vitripennis and its origin was traced by transposon similarity to the genus Trichomalopsis, which is closely related to Nasonia. To determine whether both PSR chromosomes have a similar origin we aimed to reveal the origin of the Trichogramma PSR chromosome. Using fluorescent in situ hybridization, we discovered a major satellite repeat on the PSR chromosome, the 45S ribosomal DNA. Analysis of the internal transcribed spacer 2 (ITS2) of this repeat showed the presence of multiple ITS2 sequences on the PSR chromosome resembling either the ITS2 of T. oleae or of T. kaykai. We therefore conclude that the Trichogramma PSR chromosome originates from T. oleae or a T. oleae-like species. Our results are consistent with different origins for the PSR chromosomes in Trichogramma and Nasonia.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.