利用高灵敏的TSA-FISH在玉米中定位bz1、bz2基因(英文)

植物中 ,程序性细胞死亡 (PCD)发生在植物生殖和发育的许多方面 ,已有的研究表明 ,在玉米种子的发育过程中 ,胚乳组织经历了程序性细胞死亡的过程。bz1 (bronze)和bz2是与种子的糊粉层发育相关的花青素生物合成基因 ,在玉米基因组中 ,bz1基因所在区域是重组热点 ,bz2与类黄酮的酰化、糖基化、转运、沉积等有关 ,基因的物理定位有利于基因的分离和克隆。TSA FISH (Tyramidesignalamplificationfluorescenceinsituhybridization)是一种新颖的高灵敏度的荧光原位杂交技术 ,它的主要反应原理是辣根过氧化物酶催化过氧化氢和标记的酪胺分子 (tyramide)的苯环部分反应 ,使荧光标记的酪胺分子在直接带有或间接带有HRP报告分子的探针周围沉积 ,信号因此得以极大的放大 ,从而大大提高了荧光原位杂交技术的灵敏度 ,90年代中期开始引入动物和人类组织化学和细胞遗传学研究中 ,2 0 0 1年才应用于植物细胞遗传学的研究。利用这一技术 ,我们将bz1基因定位于玉米的第 9染色体的短臂和第 1染色体的长臂上 ,其信号点距着丝粒的百分距离分别为 40 .2 ,75 .4;bz2基因定位于玉米的第 1染色体的长臂和第 5染色体的短臂上 ,其信号点距着丝粒的百分距离分别为2 1 .6,1 5 .3。本文讨论了TSA FISH技术在植物中小的、低拷贝的DNA序     

The human QARS locus: assignment of the human gene for glutaminyl-tRNA synthetase to chromosome 1q32–42

Summary We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.     

FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor.

Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Localization of the villin gene on human chromosome 2q35-q36 and on mouse chromosome 1

Summary A partial cDNA clone coding for the 110 carboxyterminal amino acids of human villin was used for mapping the human villin gene. In situ hybridization experiments on human chromosomes with tritiated probe allowed the regional localization of the villin locus to chromosome 2 at q35-36. Data obtained from restriction fragment length polymorphism analysis of two mouse species demonstrated the assignment of the villin gene to mouse chromosome 1 by assessment of linkage with the fast skeletal isoform of the myosin light-chain gene. These villin gene localizations add a fourth locus to the conserved gene cluster encoding the fast skeletal muscle isoform of the myosin light chain, isocitrate dehydrogenase, and the crystallins and confirm the partial homology of the human chromosome 2 long arm and mouse chromosome 1.     

Sublocalization of the human PDGF A-chain gene to chromosome 7, band q11.23, by in situ hybridization

The human platelet-derived growth factor A-chain (PDGFA) locus was mapped by in situ hybridization. By use of human cDNA probes encoding the PDGF A-chain precursor polypeptide the gene was assigned to the proximal long arm of chromosome 7, band q11.23. Of 76 cells with silver grains on chromosome 7, 28% had label over this band. Our assignment represents a confirmation and further sublocalization of the PDGFA locus. The location correlates with specific chromosomal abnormalities associated with certain human developmental malformations and neoplasms.     

程序性细胞死亡抑制基因Dad-1在水稻和玉米中的FISH定位(英文)

Dad-1是一种在动物和植物中都非常保守的细胞程序性死亡 (PCD) 抑制基因。作者利用 FISH (荧光原位杂交)首次把单拷贝水稻Dad-1基因物理定位在水稻第2号染色体短臂的端部(Fig.2 A,B&C)。我们还分析了它在玉米基因组中的同源序列。Southern 杂交结果显示在玉米基因组中确实存在水稻Dad-1 的同源序列(Fig.1)。FISH进一步展示了三个杂交信号分别在玉米4、5号染色体长臂和9号染色体短臂上(Fig.2 D,E&F),其信号距着丝粒的百分距离(FL值)分别为 91、98和96。其杂交位点的位置与水稻Dad-1所处的相对位置是相似的,它们都处于染色体臂的端部。这表明在一定的程度上,Dad-1基因不仅在序列同源性上而且在所处的染色体位置上具有保守性。 水稻Dad-1基因在水稻中的杂交信号检出率 (38%) 高于玉米中的。这表明与玉米相比,水稻Dad-1 基因的编码序列更容易与水稻染色体杂交;它与玉米中的相应序列可能只是部分同源。     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

Localization of histidase to human chromosome region 12q22----q24.1 and mouse chromosome region 10C2----D1

The human gene for histidase (histidine ammonia-lyase; HAL), the enzyme deficient in histidinemia, was assigned to human chromosome 12 by Southern blot analysis of human X mouse somatic cell hybrid DNA. The gene was sublocalized to region 12q22----q24.1 by in situ hybridization, using a human histidase cDNA. The homologous locus in the mouse (Hal) was mapped to region 10C2----D1 by in situ hybridization, using a cell line from a mouse homozygous for a 1.10 Robertsonian translocation. These assignments extend the conserved syntenic region between human chromosome 12 and mouse chromosome 10 that includes the genes for phenylalanine hydroxylase, gamma interferon, peptidase, and citrate synthase. The localization of histidase to mouse chromosome 10 suggests that the histidase regulatory locus (Hsd) and the histidinemia mutation (his), which are both known to be on chromosome 10, may be alleles of the histidase structural gene locus.     

Physical Localization of Genes for CaM and Ca2+- ATPase in Rice by in situ Hybridization

CaM and Ca2 + -ATPase genes are important components of signal transduction chains which affect the regulation of' gene expression and development in plants. These two genes are tunctionally closely related. The rice ( Oryza sativa L. )cDNA probes C419 and SSU304 for these two genes, which are small single-copy ones and 0.8 and 0.3 kb in size respectively, were first physically mapped on rice chromosomes by biotin-labeled in situ hybridization. Both probes were detected on chromosome 5. The detection rate was 6.18 %, and the average chromosome ann ratios and standard deviations of detected chromosomes for probes C419 and SSU304 were 1.79 ± 0.06 and 1.91 ± 0.08 respectively. Probe C419 for CaM gene was located at the end of the long ann, and probe SSU304 for Ca2 + -ATPase gene -- on the short arm near the centromere. As it was reported before, they were closely linked in the high density genetic map. This demonstrated that there was a large discrepancy between the results of genetic and physical mapping of genes, and it indicated that the region between the functionally related genes could be the cold spot. What relationship there is between the region and gene expression is to be shudied further. The nonradioactive in situ hybridization technique about the physical mapping of low/single copy, short DNA fragments is also discussed.     

Assignement of the fibronectin (FN1) gene to pongo pygmaeus (Organgutan) chromosome 11

Using Fibronectin (F18) human cDNA probe, the FN gene was assigned to orangutan chromosome 11 by in situ hybridization. Our data confirm the homology between orangutan chromosome 11 and the long arm of human chromosome 2.     

Chromosomal localization of chicken sequences homologous to the beta-actin gene by in situ hybridization

In situ DNA/chromosome hybridization techniques were used to localize the cytoplasmic beta-actin gene in the chicken. Hybridization of a beta-actin cDNA probe to metaphase chromosome spreads indicated that sequences complementary to this probe are located on the long arm of chromosome 2 (2q) and one of chromosomes 9 through 12.     

Assignment of cathepsin E (CTSE) to human chromosome region 1q31 by in situ hybridization and analysis of somatic cell hybrids

A full length cathepsin E (CTSE) cDNA clone was used to assign the corresponding gene to human chromosome region 1q31 by in situ hybridization. Southern blot analysis of DNA from three independent human x rodent somatic cell hybrids containing X;1 translocations confirmed the assignment of the CTSE gene to the distal region of the long arm of chromosome 1.     

A highly polymorphic locus in human DNA revealed by probes from cosmid 1-5 maps to chromosome 2q35----37.

The highly polymorphic locus D2S3 is revealed by three single-copy probes from cosmid C1-5. These probes, 1-30, 1-32, and 2-96, collectively reveal seven restriction fragment length polymorphisms. Fifty-three of 56 unrelated individuals (93%) were heterozygous at one or more of the seven loci, making the compound locus a very useful marker for gene mapping. Chromosomal assignment of D2S3 was obtained using a panel of human X hamster and human X mouse somatic cell hybrids. Molecular hybridization of EcoRI-digested DNA from these cell lines with the DNA inserts from subclones 1-30, 1-32, and 2-96 showed that all three probes mapped to the long arm of chromosome 2. Additionally, in situ hybridization of [3H]-labeled probe 2-96 to metaphase chromosome preparations allowed more precise assignment of the locus to the region 2q35----37.     

Assignment of the mouse desmin gene to chromosome 1 band C3

The chromosomal localization of the mouse gene coding for desmin, one of the muscle-specific intermediate filament subunits, was determined by in situ hybridization using a specific 3H-labelled DNA probe. There is only one copy of the desmin gene and it is located on chromosome 1 in the band C3. This result adds an eleventh locus to a conserved gene cluster and confirms the partial homology that exists between the long arm of human chromosome 2 and chromosome 1 of the mouse.     

A human immunoglobulin kappa orphon without sequence defects may be the product of a pericentric inversion.

The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization. The V108 region may have been transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Possible reasons for the conservation of its sequence are discussed. In spite of its bona fide sequence V108 is considered to be an unlikely candidate for a VK-JK rearrangement and subsequent functional expression.     

Assignment of the rabbit genes for alpha (PHKA) and beta (PHKB) phosphorylase kinase subunits.

The chromosomal locations of the rabbit genes for the alpha and beta subunits of phosphorylase kinase (PHKA and PHKB) were determined by in situ hybridization using rabbit cDNA probes. Our results localize PHKA to the X chromosome at the proximal end of the long arm, near the centromere, and PHKB to the same location on chromosome 5. These assignments support previously reported homoeologies of rabbit and human chromosomes.     

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.     

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome. Received: 9 January 1998