The First Knockout Mouse Model of Retinoblastoma

The retinoblastoma susceptibility gene (RB1) was the first tumor suppressor gene identified in humans (Friend, et al., 1986) and the first tumor suppressor gene knocked out by targeted deletion in mice (Jacks, et al., Clarke, et al., Lee, et al., 1992). Children with a germline mutation in one of their RB1 alleles are likely to experience bilateral multifocal retinoblastoma; however, mice with a similar disruption of Rb1 do not develop retinoblastoma. The absence of a knock-out mouse model of retinoblastoma has slowed the progress toward developing new therapies and identifying secondary genetic lesions that occur after disruption of the Rb signaling pathway. Several advances have been made, over the past several years, in our understanding of the regulation of proliferation during retinal development (Zhang, et al., 2004; Dyer J, 2004; Dyer, Cepko, 2001) and we have built upon these earlier studies to generate the first nonchimeric knock-out mouse model of retinoblastoma. These mice are being used as a preclinical model to test new therapies for retinoblastoma and to elucidate the downstream genetic events that occur after inactivation of Rb1 or its related family members.     

Molecular detection of chromosomal translocations that disrupt the putative retinoblastoma susceptibility locus.

A candidate DNA sequence with many of the properties predicted for the retinoblastoma susceptibility (RB1) locus has been cloned (S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature [London] 323:643-645, 1986). The large size of this gene (ca. 200 kilobases [kb]) and its multiple dispersed exons (Wiggs et al., N. Engl. J. Med. 318:151-157, 1988) complicate molecular screening strategies important in prenatal and presymptomatic diagnosis and in carrier detection. Here we used field inversion gel electrophoresis (FIGE) to construct a restriction map of approximately 1,000 kb of DNA surrounding the RB1 locus and to detect the translocation breakpoints in three retinoblastoma patients. DNA probes from either the 5' or 3' end of the gene were used to detect a 250-kb EagI restriction fragment in DNA from unaffected individuals. Both probes identified an additional hybridizing fragment in the DNA from each patient, permitting the breakpoints in all three to be mapped within the cloned RB1 gene. Analysis of the breakpoint in one translocation cell line allowed the RB1 gene to be oriented with its 5' end toward the centromere. The 5' end of the gene also appeared to be associated with a clustering of sites for several infrequently cleaving restriction enzymes, indicating the presence of an HpaII tiny fragment island. The detection and mapping of the translocation breakpoints of all three retinoblastoma patients to within the putative RB1 gene substantiated the authenticity of this candidate sequence and demonstrated the utility of FIGE in detecting chromosomal rearrangements affecting this locus.     

贵州竹子新报道

发表了产于贵州的玉山竹属2新种,即窄叶玉山竹和细弱玉山竹。赤竹（Sasa longiligula McClure）、单枝竹（Bonia saxatilis(Chia, H. L. Fung et Y. L. Yang) N. H. Xia）为贵州新分布竹种,并对其中赤竹的花做了补充描述。     

Template switching: from replication fork repair to genome rearrangements

Genome rearrangements are a hallmark of human genomic disorders and occur largely through recombination mechanisms. In this issue, Lee et al. (2007) show that the complex nonrecurrent rearrangements observed in the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD) are likely to be caused by a replication mechanism involving template switching.     

Human retinoblastoma gene: long-range mapping and analysis of its deletion in a breast cancer cell line.

Mutational inactivation of the retinoblastoma (RB) gene is considered a fundamental event in the formation of several types of human cancer. A substantial proportion of RB gene mutations are partial or complete deletions that extend an unknown distance beyond one or both ends of the gene. To provide a framework for measuring the extent of these deletions, we have constructed a long-range restriction map of SfiI sites spanning 850 kilobases around the RB gene. This map was applied in a molecular analysis of RB gene deletion in breast cancer cell line MB468. A previous study of this cell line demonstrated deletion of the entire RB gene except for exons 1 and 2 (E. Y.-H. P. Lee, H. To, J.-Y. Shew, R. Bookstein, P. Scully, and W.-H. Lee, Science 241:218-221, 1988). Genomic clones containing the deletion junction were isolated from a library made from MB468 DNA. A probe obtained from the far side of the deletion junction was used to localize and clone the unknown 3' endpoint, demonstrating that the chromosomal mutation in this case was a simple deletion spanning 200 kilobases. Sequence analysis of the deletion junction indicated a conservative deletion with no loss or gain of nucleotides. The deletion endpoints had no sequence homology to each other or to any repetitive sequence family, such as Alu, so the recombination event was illegitimate. Structural analysis of this and other RB gene deletions is important for understanding molecular mechanisms of recessive oncogenesis.     

中国(竹思)簩竹属(Schizostachyum Nees)的研究及其它

对国产(竹思)(竹劳)竹属(Schizostachyum Nees)进行了系统整理。对属的范围进行了修订,认为本属应包括乔草竹属(Dendrochloa Parkinson),薄竹属(Leptocanna Chia et H.L.Fung),长穗竹属(Teinostachyum Munro)和李海竹属(Neohouzeaua A.Camus),并首次建立了本属的属下分类系统。此外,对二种竹子起了新名:甲竹(Bambusa austro-sinensis Xia)和毛环单竹(B.yunnanensis Xia);作了4个新组合,即Schizostachyum coradatum(Wen et Dai)Xia, S.dumetorum(Hance)Munro var.xinwuense(Wen et J. Y Chin)Xia, Bambusa glaucescens glaucescens(Willd)Sieb. ex Munro var.annulata(W.T.Lin et Z.J.Feng)Xia和B.glaucescens (Willd)Sieb.ex Munro var.pubivagina(W.T.Lin et Z.J.Feng)Xia.     

H3K27 demethylases, at long last

Methylation of lysine 27 on histone H3 (H3K27me) by the Polycomb complex (PRC2) proteins is associated with gene silencing in many developmental processes. A cluster of recent papers (Agger et al., 2007; De Santa et al., 2007; Lan et al., 2007; Lee et al., 2007) identify the JmjC-domain proteins UTX and JMJD3 as H3K27-specific demethylases that remove this methyl mark, enabling the activation of genes involved in animal body patterning and the inflammatory response.     

H2AX haploinsufficiency modifies genomic stability and tumor susceptibility

Histone H2AX becomes phosphorylated in chromatin domains flanking sites of DNA double-strand breakage associated with gamma-irradiation, meiotic recombination, DNA replication, and antigen receptor rearrangements. Here, we show that loss of a single H2AX allele compromises genomic integrity and enhances the susceptibility to cancer in the absence of p53. In comparison with heterozygotes, tumors arise earlier in the H2AX homozygous null background, and H2AX(-/-) p53(-/-) lymphomas harbor an increased frequency of clonal nonreciprocal translocations and amplifications. These include complex rearrangements that juxtapose the c-myc oncogene to antigen receptor loci. Restoration of the H2AX null allele with wild-type H2AX restores genomic stability and radiation resistance, but this effect is abolished by substitution of the conserved serine phosphorylation sites in H2AX with alanine or glutamic acid residues. Our results establish H2AX as genomic caretaker that requires the function of both gene alleles for optimal protection against tumorigenesis.     

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).     

云南及近邻地区竹亚科植物新记录

报道了云南竹亚科新记录种 5种 1变种 ,即吊丝竹 (Dendrocalamus minor(Mc Clure) Chia et H.L.Fung ) ,花吊丝竹 (Dendrocalamus minor var. amoenus(Q. H. Dai et C. F. Huang) Hsueh et D. Z. Li) ,黔竹(Dendrocalamus tsiangii(Mc Clure) Chia et H. L. Fung) ,马甲竹 (Bambusa tulda Roxb.) ,坭竹 (Bambusa gibbaMc Clure)和料慈竹 (Bambusa distegia(Keng et Keng f.) Chia et H.L.Fung)。后两者亦分别为贵州、广西的地理分布新记录种。同时 ,根据对标本和种植于 ' 99昆明世博会竹园的活竹丛的研究 ,讨论了毛环竹亦即毛环单竹 (Bambusa yunnanensis N. H. Xia)的分类学问题 ,将其归并到料慈竹 (Bambusa distegia(Keng et Keng f.) Chiaet H.L.Fung)中。     

Revision on Cephalostachyum Munro (Poaceae: Bambusoideae) in China

Species of Cephalostachyum Munro (Poaceae: Bambusoideae) from China are distributed in Yunnan and Tibet, mainly in Yunnan. In this paper, we discussed species of Cephalostachyum and compiled a key to species from China, based on recent studies on micromorphological characters of leaf epidermis and molecular phylogenetics of paleotropical woody bamboos. There is a total of seven species of Cephalostachyum distributed in China, all in Yunnan: Cchinense (Rendle) DZ. Li et HQ. Yang, Cfuchsianum Gamble et Hook. f., Cmannii (Gamble) Stapleton et DZ. Li, Cpallidum Munro, Cpingbianense (Hsueh et YM. Yang ex Yi et al.) DZ. Li et HQ. Yang, Csanguineum (WP. Zhang) DZ. Li et HQ. Yang and Cscandens Bor. Leptocanna Chia et HL. Fung and Cvirulentum YM Yang et F. Du are synonyms of Cephalostachyum Munro and Cfuchsianum Gamble et Hook. f. respectively. On the other hand, Cpergracile Munro and Cvirgatum (Munro) Kurz are morphologically closer to Schizostachyum Nees than to Cephalostachyum, and they should be treated as members of Schizostachyum. This paper is of significance to a worldwide revision of Cephalostachyum.     

Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus

The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).