乙肝相关性肝癌差异表达基因的获得及FOLR1分析

运用基因芯片技术获取以稳定转染HBx基因的肝癌细胞HepG2(HepG2-X)及非转染的肝癌细胞HepG2中差异表达的基因,并对其中一条基因进行的生物信息学分析。采用人肝癌G2细胞(HepG2)细胞系为对照组,以稳定转染HBx的HepG2细胞为实验组,抽取总RNA,经过反转录cDNA,对照组用Cy3实验组用Cy5荧光标记,获得cDNA探针;经杂交、洗涤后,通过ImaGene3.0软件进行分析统计。通过基因芯片筛选,获得643条与乙肝相关性肝细胞性癌相关的基因,其中FOLR1基因差异性表达最显著,HBx显著下调其表达,同源性比较分析结果表明,其碱基序列与已经报道的其他12种哺乳动物的相似率为67%-99%,且符合种属之间的进化关系。基因芯片筛选HBx诱导的HepG2差异表达基因具有样品用量少,高质量,高速度,高敏感等特性。FOLR1可能为HBV相关肝癌的发生、转移的诊断、靶基因治疗和预后评估提供一定的依据。     

The T-box repressors TBX2 and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in the initiator

The murine tumor suppressor p19(ARF) (p14(ARF) in humans) is thought to fulfill an important protective role in preventing primary cells from oncogenic transformation via its action in the p53 pathway. Several disease-implicated regulators of p19(ARF) are known to date, among which are the T-box genes TBX2, which resides on an amplicon in primary breast tumors, and TBX3, which is mutated in the human developmental disorder Ulnar-Mammary syndrome. Here we identify a variant T-site, matching 13 of 20 nucleotides of a consensus T-site, as the essential TBX2/TBX3-binding element in the human p14(ARF) promoter. Mutant analysis indicates that both the consensus T-box and a C-terminal conserved repression domain are essential for p14(ARF) repression. Whereas the core nucleotides required for interaction of the archetypal T-box protein Brachyury with a consensus T-site are conserved in the variant site, additional flanking nucleotides contribute to the specificity of TBX2 binding. This is illustrated by the inability of TBX1A or Xbra to activate via the variant p14(ARF) T-site. Importantly, this suggests a hitherto unsuspected level of specificity associated with T-box factors and corresponding recognition sites in regulating their target genes in vivo.     

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1.

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-beta family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-beta superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.     

The mtDNA genealogy of closely related Drosophila silvestris.

Genetic, morphological, and behavioral analyses have been used to examine the evolutionary dynamics and phylogeny of the rare Hawaiian Drosophila species, D. silvestris. Critical to understanding the evolution of this species is the examination of the distribution of populations of D. silvestris on the Big Island of Hawaii. Behavioral analysis using mating asymmetries and the Kaneshiro hypothesis as an indicator of ancestral behavioral state has suggested that flies from the northern part of the island are ancestral to those on the southern part of the island. Consequently, a sequential pattern of colonization going from north to south is predicted for these flies on the east side of the Island of Hawaii. We have examined this prediction using mitochondrial DNA (mtDNA) restriction site analysis with four-base cutters and DNA sequencing. The resulting mtDNA phylogeny based on 23 phylogenetically informative restriction sites and two phylogenetically informative DNA sequence characters agrees in part with the phylogeny predicted from the behavioral data.     

Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1

To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we performed representational difference analysis (RDA) using 32Dcl3 myeloblastic cells that were deprived of IL-3 for 24h. We have isolated a novel cDNA (g1-related protein, G1RP) that is homologous to g1, a Drosophila melanogaster zinc-finger protein that is expressed in the mesoderm. Northern blot analysis using RNAs derived from 32Dcl3 cells that have been grown in the absence of IL-3 demonstrates that the G1RP message is upregulated in these cells following the removal of IL-3, suggesting that this gene may regulate growth factor withdrawal-induced apoptosis of myeloid precursor cells.     

Hybridization between closely related songbirds is related to human habitat disturbance

Human habitat disturbances can promote hybridization between closely related, but typically reproductively isolated, species. We explored whether human habitat disturbances are related to hybridization between two closely related songbirds, black-capped and mountain chickadees, using both genomic and citizen science data sets. First, we genotyped 409 individuals from across both species' ranges using reduced-representation genome sequencing and compared measures of genetic admixture to a composite measure of human landscape disturbance. Then, using eBird observations, we compared human landscape disturbance values for sites where phenotypically diagnosed hybrids were observed to locations where either parental species was observed to determine whether hybrid chickadees are reported in more disturbed areas. We found that hybridization between black-capped and mountain chickadees positively correlates with human habitat disturbances. From genomic data, we found that (1) hybrid index (HI) significantly increased with habitat disturbance, (2) more hybrids were sampled in disturbed habitats, (3) mean HIs were higher in disturbed habitats versus wild habitats, and (4) hybrids were detected in habitats with significantly higher disturbance values than parentals. Using eBird data, we found that both hybrid and black-capped chickadees were significantly more disturbance-associated than mountain chickadees. Surprisingly, we found that nearly every black-capped chickadee we sampled contained some proportion of hybrid ancestry, while we detected very few mountain chickadee backcrosses. Our results highlight that hybridization between black-capped and mountain chickadees is widespread, but initial hybridization is rare (few F1s were detected). We conclude that human habitat disturbances can erode pre-zygotic reproductive barriers between chickadees and that post-zygotic isolation is incomplete. Understanding what becomes of recently hybridizing species following large-scale habitat disturbances is a new, but pressing, consideration for successfully preserving genetic biodiversity in a rapidly changing world.     

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless.

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn-linked glycosylation sites. The protein sequence shows marked similarity with human and murine int-1 and their Drosophila homolog wingless (Dint-1). This human int-1 related protein, int-1 and Dint-1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.     

Isolation from the rumen of a new acetogenic bacterium phylogenetically closely related to Clostridium difficile

Five strains of filamentous acetogenic bacterium were isolated from high dilutions of ruminal content of newborn lambs. These Gram-positive spore-forming bacteria grew either chemolithotrophically with H2+ CO2 or chemo-organotrophically with glucose, cellobiose, fructose, maltose, mannose and syringic acid. The DNA base composition of the five strains were between 29.1 and 31.3 mol% G + C. Their temperature and pH optimum for growth were 35-40 degrees C and 6.5-7.0, respectively. The full 16S rRNA gene sequence analysis of the reference strain indicated that it was most closely related to Clostridium difficile. The sequence similarity value between the 16S rRNA gene of the reference strain and this pathogenic strain was 99.7%.     

Dissecting contiguous gene defects: TBX1

DiGeorge syndrome is mainly caused by a multigene, heterozygous, interstitial chromosomal deletion. Of the approximately 30 deleted genes, Tbx1 is the only gene that, after an extensive functional analysis in the mouse, has been found to be haploinsufficient. The mutant phenotype is convincingly similar to the human syndrome, and its human homolog, TBX1, is the only gene for which mutations have been found in some patients without the chromosomal deletion. The research interest in this syndrome is driven not only by the obvious clinical significance of the disease but also by a broader biological importance. In particular, this syndrome is the most typical developmental defect of the embryonic pharyngeal system: a transient, vertebrate-specific structure that contributes to diverse tissues of the head, neck and thorax. Many birth defects, including a large fraction of congenital heart disease cases, derive from developmental problems of the pharyngeal system. Tbx1 is an excellent tool to probe the genetic network governing embryonic pharyngeal development.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.     

Apis mellifera cytoplasmic elongation factor 1 alpha (EF-1 alpha) is closely related to Drosophila melanogaster EF-1 alpha

Using low stringency hybridisation with a Drosophila melanogaster EF-1 alpha gene fragment we have isolated a genomic DNA clone encoding elongation factor 1 alpha (EF-1 alpha) from Apis mellifera. The hybridising Apis mellifera sequence could be delineated to two small EcoRI fragments that were also revealed by genomic Southern hybridisation. By comparison with the corresponding Drosophila melanogaster data the complete translational reading frame has been deduced. It is interrupted by two intervening sequences of 220 and about 790 nucleotides. Comparison with known eucaryotic EF-1 alpha sequences further confirms that certain amino acid sequences seem to be invariable within the EF-1 alpha protein family.