Interferon and c-ets-1 genes in the translocation (9;11)(p22;q23) in human acute monocytic leukemia

Gene probes for interferons alpha and beta 1 and v-ets were hybridized to metaphase chromosomes from three patients with acute monocytic leukemia who had a chromosomal translocation, t(9;11)(p22;q23). The break in the short arm of chromosome 9 split the interferon genes, and the interferon-beta 1 gene was translocated to chromosome 11. The c-ets-1 gene was translocated from chromosome 11 to the short arm of chromosome 9 adjacent to the interferon genes. No DNA rearrangement was observed when these probes were hybridized to genomic DNA from leukemic cells of two of the patients. The results suggest that the juxtaposition of the interferon and c-ets-1 genes may be involved in the pathogenesis of human monocytic leukemia.     

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.     

The role of the c-mos gene in the 8;21 translocation in human acute myeloblastic leukemia

The human c-mos proto-oncogene is located on chromosome 8 at band q22, close to the breakpoint in the t(8;21) (q22;q22) chromosome rearrangement. This translocation is associated with acute myeloblastic leukemia, subgroup M2. The c-myc gene, another proto-oncogene, has been mapped to 8q24. The breakpoint at 8q22 separates these genes, as determined by in situ hybridization of c-mos and c-myc probes. The c-mos gene remains on the 8q-chromosome and the c-myc gene is translocated to the 21q+ chromosome. Southern blot analysis of DNA from bone marrow cells of four patients with this translocation showed no rearrangement of c-mos.     

The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining

In this study, the joining sequences between chromosomes 14 and 18 on the 14q+ chromosomes of a patient with pre-B-cell leukemia and four patients with follicular lymphoma carrying a t(14;18) chromosome translocation were analyzed. In each case, the involved segment of chromosome 18 has recombined with the immunoglobulin heavy-chain joining segment (JH) on chromosome 14. The sites of the recombination on chromosome 14 are located close to the 5' end of the involved JH segment, where the diversity (D) regions are rearranged with the JH segments in the production of active heavy-chain genes. As extraneous nucleotides (N regions) were observed at joining sites and specific signal-like sequences were detected on chromosome 18 in close proximity to the breakpoints, it is concluded that the t(14;18) chromosome translocation is the result of a mistake during the process of VDJ joining at the pre-B-cell stage of differentiation. The putative recombinase joins separated DNA segments on two different chromosomes instead of joining separated segments on the same chromosome, causing a t(14;18) chromosome translocation in the involved B cells.     

Beta amyloid gene duplication in Alzheimer's disease and karyotypically normal Down syndrome

With the recently cloned complementary DNA probe, lambda Am4 for the chromosome 21 gene encoding brain amyloid polypeptide (beta amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this gene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzheimer's disease.     

植物流式细胞遗传学及其应用研究进展*

 流式细胞遗传学是利用流式细胞术对植物有丝分裂细胞的染色体进行分离、纯化和分选等操作的细胞遗传学分支学科,已在许多领域广泛的应用,如与DNA 分子标记结合进行物理作图,利用FISH和PRINS进行细胞遗传作图,重组DNA文库的构建,标记片段的定向分离以及蛋白质分析。利用流式细胞技术已对数十种植物物种进行了染色体分析,应用最多的是豆类作物和禾谷类作物。流式细胞术有许多潜在的利用价值,主要包括植物染色体和染色体臂特异BAC文库的构建,低拷贝序列的目标分离,ESTs和杂交DNA 序列的高产量作图以及对染色体蛋白的整体分析等。本文综述了植物流式细胞遗传学的研究方法及其在植物研究中的应用,并对其在植物基因组分析的潜在利用价值作了展望。     

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.     

基于基因组原位杂交快速构建黄瓜变种间核型

【目的】利用基因组荧光原位杂交（genomic in situ hybridization,GISH）技术,对黄瓜（Cucumis sativus L.,2n=2x=14）种内两个变种（栽培黄瓜C. sativus var. sativus和野生黄瓜C. sativus var hardwickii）进行中期染色体分析,建立黄瓜变种染色体核型的快速分析方法,为黄瓜细胞分子遗传学研究提供基础。【方法】以栽培黄瓜‘9930’和野生黄瓜C. sativus var. hardwickii为材料,利用CTAB法提取栽培黄瓜‘9930’的基因组总DNA,采用缺刻平移法,将栽培黄瓜‘9930’基因组DNA和45S rDNA分别利用地高辛和生物素标记为探针,与栽培黄瓜‘9930’和野生变种C.sativus var. hardwickii的中期染色体进行荧光原位杂交,根据杂交结果显示的栽培黄瓜与野生变种每条染色体GISH荧光带型的不同,结合45S rDNA位点信号特征,区分栽培黄瓜与野生变种的每条染色体,并进行核型分析。【结果】荧光原位杂交结果显示,GISH信号并非平均分布于所有染色体上,而是在不同染色体的特定部位产生独特的信号,且两个变种间中期染色体的GISH信号模式差异显著。在栽培黄瓜‘9930’有丝分裂中期染色体上,除了6号染色体仅在短臂末端和近着丝粒处产生GISH信号外,其他染色体上的GISH信号集中分布于染色体的两端和近着丝粒的一侧或两侧,且每条染色体的信号特征差异明显;45S rDNA信号主要分布于‘9930’的第1、2、3、4和7号染色体的近着丝粒处,有3对强信号和2对弱信号。在野生黄瓜C. sativus var. hardwickii有丝分裂中期染色体上,杂交信号的位置及强弱与栽培黄瓜‘9930’表现明显不同,近着丝粒处均有GISH信号,但仅在第1、2、4和5号染色体的一端产生GISH信号,45S rDNA信号仅出现在第1、2和3号染色体上,表现为第1号染色体上信号极强,第2和3号染色体上信号极微弱。这些结果显示,以栽培黄瓜基因组DNA为探针的荧光原位杂交能反应出两个变种中期染色体独特的信号分布模式,通过信号的分布模式和强弱,结合45S rDNA位点信号的特异分布,可对每条染色体进行清晰地鉴别,并据此建立了两个变种的核型模式。比较前人发表的黄瓜已有重复序列的分布图,发现GISH揭示的信号分布主要位于黄瓜染色体串联重复序列区域。【结论】黄瓜基因组原位杂交能一次性快速显示基因组串联重复序列的分布图,能有效地用于不同黄瓜变种的快速核型分析;同时发现染色体上串联重复序列的分布及强弱在黄瓜变种间表现出明显的分化。     

甘蓝2号染色体的高分辨率5S rDNA荧光原位杂交

 【目的】羽衣甘蓝5S rDNA 的染色体定位和拷贝数分析,为进一步利用FISH进行2号染色体基因定位和细胞遗传图谱构建奠定基础。【方法】以羽衣甘蓝为材料,采用荧光原位杂交技术将DIG标记的5S rDNA探针定位于不同分辨率的绒毡层细胞中期染色体、粗线期染色体以及伸长DNA纤维上。【结果】在中期染色体和粗线期染色体上,都同时获得3个杂交信号位点（a、b、c）,且位于2号染色体的长臂近着丝粒区域,其信号强度为b＞a＞c;而在伸长DNA纤维上,出现了3种不同长度的念珠状长链（a、b、c）, 其物理大小分别为257、359和134 kb,这3种长链分别与3个信号位点形成一一对应关系。【结论】在羽衣甘蓝2号染色体上存在3个串联重复位点,粗略估算出3个5S rDNA位点的拷贝数分别为510、712和266。     

Common pathogenetic mechanism for three tumor types in bilateral acoustic neurofibromatosis

Bilateral acoustic neurofibromatosis (BANF) is a genetic defect associated with multiple tumors of neural crest origin. Specific loss of alleles from chromosome 22 was detected with polymorphic DNA markers in two acoustic neuromas, two neurofibromas, and one meningioma from BANF patients. This indicates a common pathogenetic mechanism for all three tumor types. The two neurofibromas were among three taken from the same patient, and both showed loss of identical alleles demonstrating that the same chromosome suffered deletion in both tumors. The third neurofibroma from this patient showed no detectable loss of heterozygosity, which suggests the possibility of a more subtle mutational event that affects chromosome 22. In the two acoustic neuromas, only a portion of chromosome 22 was deleted, narrowing the possible chromosomal location of the gene that causes BANF to the region distal to the D22S9 locus in band 22q11. The identification of progressively smaller deletions on chromosome 22 in these tumor types may well provide a means to clone and characterize the defect.     

黑麦染色体组中一个新重复序列的发现、定位与应用

 【目的】寻找黑麦基因组中新的重复序列作为特异PCR标记。【方法】以普通小麦中国春、川农18、育成品系R111、绵阳11为对照，以荆州黑麦、秦岭黑麦、非洲黑麦、森林黑麦为材料，用RAPD法筛选到黑麦基因组中的一个高拷贝DNA片段OPD15940，将OPD15940输入到NCBI的BLAST框中进行比对。根据OPD15940设计特异PCR引物D15F和D15R，利用这对引物对小麦族物种进行扩增，验证OPD15940的特异性。进而利用原位杂交技术定位pScD15940在染色体上的位置。【结果】序列比对后发现OPD15940与重复序列Sukkula中近60个53bp的小片段有较高的同源性，但又不同于Sukkula，是一类新的重复序列。通过特异PCR确定仅含黑麦染色质的物种能扩增出OPD15940，因而OPD15940为黑麦所特有。原位杂交结果显示除端部区域外，pScD15940弥散状分布在黑麦整套染色体上。【结论】OPD15940可以作为分子标记检测导入到小麦背景中的黑麦染色体。     

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) was identified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes were microisolated and collected. After two rounds of PCR amplification, the PCR products were ranged from 150 - 3 000 bp,with predominant fragments at about 200 - 2 000 bp. Using Ag.intermediumgenomic DNA as a probe, Southern blotting analysis confirmed the products originated from Ag. intermediumgenome. The products were purified, ligated to pUC18 and then transformed into competence E.coli DH5α to produce a 2Ai-2 chromosome DNA library. The microcloning experiments produced approximately 5×105 clones, the size range of the cloned inserts was 200- 1 500 bp, with an average of 580bp. Using Ag. intermediumgenomic DNA as a probe, dot blotting results showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences in the library. Four Ag. intermedium clones were screened from the library by RFLP, and three clones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitive sequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitive DNA sequence.     

The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites

Fragile X syndrome is a common form of mental retardation associated with a fragile site on the human X chromosome. Although fragility at this site is usually evident as a nonstaining chromatid gap, it remains unclear whether or not actual chromosomal breakage occurs. By means of somatic cell hybrids containing either a normal human X or a fragile X chromosome and utilizing two genes that flank the fragile site as markers of chromosome integrity, segregation of these markers was shown to be more frequent if they encompass the fragile site under appropriate culture conditions. Hybrid cells that reveal marker segregation were found to contain rearranged X chromosomes involving the region at or near the fragile site, thus demonstrating true chromosomal breakage within this area. Two independent translocation chromosomes were identified involving a rodent chromosome joined to the human X at the location of the fragile site. DNA analysis of closely linked, flanking loci was consistent with the position of the breakpoint being at or very near the fragile X site. Fragility at the translocation junctions was observed in both hybrids, but at significantly lower frequencies than that seen in the intact X of the parental hybrid. This observation suggests that the human portion of the junctional DNA may contain part of a repeated fragility sequence. Since the translocation junctions join heterologous DNA, the molecular cloning of the fragile X sequence should now be possible.     

The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.     

Genomic and genetic definition of a functional human centromere

The definition of centromeres of human chromosomes requires a complete genomic understanding of these regions. Toward this end, we report integration of physical mapping, genetic, and functional approaches, together with sequencing of selected regions, to define the centromere of the human X chromosome and to explore the evolution of sequences responsible for chromosome segregation. The transitional region between expressed sequences on the short arm of the X and the chromosome-specific alpha satellite array DXZ1 spans about 450 kilobases and is satellite-rich. At the junction between this satellite region and canonical DXZ1 repeats, diverged repeat units provide direct evidence of unequal crossover as the homogenizing force of these arrays. Results from deletion analysis of mitotically stable chromosome rearrangements and from a human artificial chromosome assay demonstrate that DXZ1 DNA is sufficient for centromere function. Evolutionary studies indicate that, while alpha satellite DNA present throughout the pericentromeric region of the X chromosome appears to be a descendant of an ancestral primate centromere, the current functional centromere based on DXZ1 sequences is the product of the much more recent concerted evolution of this satellite DNA.     

Fragile sites at 16q22 are not at the breakpoint of the chromosomal rearrangement in AMMoL

There is much speculation about fragile sites on human chromosomes predisposing to specific chromosome rearrangements seen in cancer. Acute myelomonocytic leukemia is characterized by neoplastic chromosome rearrangements involving band 16q22 in patients who carry the rare fragile site at 16q22. This specific leukemic breakpoint is within the metallothionein gene cluster, which is here shown to be proximal to the rare fragile site (FRA16B) and to a common fragile site (FRA16C) in this region. Hence neither of these fragile sites are at the breakpoint in this leukemic chromosomal rearrangement.