Chromosomal and nuclear distribution of the HindIII 1.9-kb human DNA repeat segment

A human interspersed repetitive DNA cloned in pBR322, the HindIII 1.9-kb (kilobase pair) sequence, was labeled with biotinylated dUTP and hybridized to acid-fixed chromosomes and paraformaldehyde-fixed whole cells in situ. Using our most sensitive detection techniques this probe highlighted on the order of 200 discrete loci, in punctate or banded arrays, that resembled a Giemsa-dark band pattern on chromosome arms. Interphase cells also displayed many discrete punctate spots of hybridization along chromosome fibers. The ubiquitous Alu sequence repeat also appeared to be concentrated in specific regions of the chromosome and predominantly highlighted Giemsa-light bands. Centromeric or ribosomal spacer DNA repeats used as controls in all studies gave the expected hybridization profiles and showed no non-specific labeling of chromosome arms. Cohesive groups of centromeric DNA arrays and rDNA clusters were observed in interphase nuclei. Refinements in methods for detecting biotin-labeled probes in situ were developed during these studies and calculations indicated that about 20 kb or more of the 1.9-kb repeat were present at each hybridization site. The chromosomal distribution of the 1.9-kb repeat suggests that this sequence may reflect, or participate in defining, ordered structureal domains along the chromosome.     

Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector

The construction of a yeast artificial chromosome containing a human DNA insert is reported. This molecule of about 200 kb behaves as a native yeast chromosome since it has a very high mitotic stability and is present in the yeast transformant clone at a copy number similar to that of the resident chromosomes. Hybridization with the TTAGGG sequence demonstrates that this chromosome contains human telomeric sequences. In situ hybridization of the biotin-labelled artificial chromosome to metaphase human chromosomes shows that the insert occupies a telomeric position on the long arm of chromosome 9. Since the fragment was cloned as an EcoRI insert and not as a telomere, it is situated medially to the telomeric sequences and harbours telomere-associated sequences, that have been shown to contain the TTAGGG sequence. The fragment represents the end of the genetic map of chromosome 9 and thus can be used to characterize the sequence and the structure of the chromosomal region that runs from the end of the chromosome to the first gene.     

The organization of two related subfamilies of a human tandemly repeated DNA is chromosome specific

Summary Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1 kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).     

Huntington disease-linked restriction fragment length polymorphism localized within band p16.1 of chromosome 4 by in situ hybridization.

A 5.5-kilobase (kb) single sequence DNA fragment (G8) reveals the DNA polymorphic locus D4S10 on Southern blot analysis. This locus is closely linked to Huntington disease and has been mapped to chromosome 4 short arm using human-mouse somatic cell hybrids, and specifically to chromosome 4 band p16 using DNA from individuals with deletions of chromosome 4 short arm who exhibit Wolf-Hirschhorn syndrome. With in situ hybridization techniques, we have confirmed the location of D4S10 on chromosome 4 and further localized it within band p16 utilizing five patients, four with overlapping chromosome 4 short-arm aberrations. The DNA segment G8 was hybridized to the mataphase chromosomes of the five patients. Two of them have different interstitial deletions of one of the chromosome 4 short arms (TA and BA), two have different chromosome 4 short-arm terminal deletions (RG and DQ), and one has a normal male karyotype. By noting the presence or absence of hybridization to the partially deleted chromosomes with known precise breakpoints, we were able to more accurately localize probe G8 to the distal half of band p16.1 of chromosome 4.     

Use of tyramide-fluorescence in situ hybridization and chromosome microdissection for ascertaining homology relationships and chromosome linkage group associations in oats

The physical mapping of single locus sequences by tyramide-fluorescence in situ hybridization (Tyr-FISH) and the analysis of sequences obtained from microdissected chromosomes were assayed as potential tools for (1) determining homology and homoeology among chromosome regions of Avena species, and (2) establishing associations between linkage groups and specific chromosomes. Low copy number probes, derived from resistance gene analogues (RGAs) and 2.8-4.5 kb long, successfully produced hybridization signals on specific chromosomes. Four sets of homoeologous chromosome regions were identified in the hexaploids using 3 probes that produced 4 single locus markers in A. strigosa and 2 in A. eriantha. Laser capture microdissection of metaphase I cells of A. sativa monosomic lines allowed the isolation of critical univalents. Sequences derived from 2 RGAs were successfully amplified in DNA extracted from univalents. In one instance, it was possible to map a nucleotide polymorphism specific for 1 chromosome. An association was established between this chromosome and its linkage groups in 2 hexaploid genetic maps. The results indicate that Tyr-FISH is useful in the characterization of homoeologous chromosome segments in hexaploids, whereas chromosome microdissection, as employed in this work, needs to be improved before it can routinely be used with meiotic chromosomes.     

A polymorphic DNA marker genetically linked to congenital malformations

Unusual restriction fragments were detected by DNA blot hybridization with PCNA (DNA polymerase-delta auxiliary protein) probe in one of seven cases of congenital malformations. Chromosomal in situ hybridization localized PCNA gene to region q31-35 of human chromosome 2. To discover the locus more closely associated with congenital malformations, a cloned DNA segment which has been mapped to chromosomal region 2q33-36 was tested for restriction fragment length polymorphisms (RFLPs) in these patients. The 2q33-36 probe hybridized with 2.1-kb, 1.9-kb and 1.7-kb fragments in ten normal control samples. In seven cases of congenital malformations examined, however, the band of 2.1 kb is absent in six cases and the band of 1.7 kb in one case. These results indicate that the locus closely linked to congenital malformations is present in the proximity of PCNA locus.     

Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cell defect in DNA replication and cell cycle progression to Xp11.2----p11.4

The temperature-sensitive ts A1S9 mouse L-cell mutant is defective in an X-linked gene essential for progression of cells through the S phase of the cell division cycle. A single copy fragment derived from the complementing human A1S9 gene was used as a probe to localize the gene on the X chromosome. Southern blot analysis of human x rodent hybrids and in situ hybridization to human metaphase chromosomes allowed the regional assignment of the human A1S9 gene to Xp11.2----p11.4.     

Chromosomal localization of human endogenous retroviral element ERV1 to 18q22----q23 by in situ hybridization

From a clone containing the entire locus of human endogenous retroviral element ERV1, we have obtained a DNA probe that is specific for the 3' long terminal repeat (LTR) sequence. This probe was used to map the LTR of ERV1 by in situ hybridization to chromosomes from normal human blood lymphocytes. The LTR was found to be localized to the distal portion of the long arm of human chromosome 18, within bands q22----q23. This chromosome locus is near the constitutive fragile site at band q21.3 on chromosome 18 associated with the 14;18 translocations seen in follicular lymphomas.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

A minisatellite with fold-back structure is included in the 5'-flanking region of the Adh gene of Scaptodrosophila lebanonensis

A tandem repetitive sequence with a repeat unit of 12 bp has been found 1.3 kb upstream of the Adh gene of Scaptodrosophila lebanonensis. This repetitive sequence extends over 4.3 kb and consists of two inverted arrays (a fold-back segment). The repeated unit with a consensus sequence GAATACAGAATA is highly conserved and the nucleotide substitutions are not distributed randomly among the 12 bp. In situ hybridization in S. lebanonensis polytene chromosomes revealed two signals, one at the 60A section, the Adh locus, and a second site in the same chromosome at the 60C section close to the telomere. This same pattern of hybridization is obtained in all the analyzed strains including the subspecies S. lebanonensis casteeli. The minisatellite sequence accounts for about 0.03-0.04% of the S. lebanonensis genome and showed intraspecific variability in tandem repeat numbers. Possible functions of this sequence are discussed.     

YAC mapping by FISH using Alu-PCR-generated probes.

Human genomic mapping has been greatly advanced by the independent development of three new methods: large DNA fragment cloning in yeast artificial chromosomes, amplification from complex DNAs of human specific segments by Alu-PCR, and high-resolution localization of complex DNA probes by fluorescent in situ hybridization. We describe here the combination of these three analytical tools for efficient and accurate localization of randomly screened or especially selected human YAC recombinants to chromosome 11. We map a YAC clone encompassing the pepsinogen A (PGA) locus to 11q13.1-11q13.3.     

ZFX基因同源序列在黄鳝基因组中的检出及其染色体定位

以大熊猫锌指蛋白基因Zfx为探针 ,在黄鳝基因组DNA中检测到一条长约 9 5kb的杂交带。依据哺乳类和爬行类动物锌指蛋白基因 (ZFX/Zfc)编码第 7～ 13个锌指结构的DNA序列保守性设计引物 ,在黄鳝基因组DNA中仅扩增到一条 5 12bp的DNA片段。将此片段克隆至载体 pBS中 ,从雌性、雄性个体中分别挑选 4个含有插入片段的白色克隆进行测序。测序结果表明 ,这些克隆中插入片段的核苷酸序列一致。该DNA片段在核苷酸水平上与人类ZFX和ZFY分别具有 88%和 87%同源性 ,但其与美洲鳄鱼Zfc的同源性可达 90 % ,而在氨基酸水平上则分别存在 95 9%、95 9%和 93 5 %的同源性 (170个氨基酸 )。该基因命名为黄鳝锌指蛋白基因Zfa ,并运用FISH将其定位于黄鳝 1号染色体 ,距离着丝粒的相对位置为 6 0 1± 0 38。通过进一步研究证明 ,黄鳝 1号染色体上存在有真兽类哺乳动物X染色质同源的保守片段 ,该保守片段有可能就是哺乳动物X染色体起源和进化的原始物质基础之一。应用哺乳动物X染色体连锁的其他基因在鱼类开展染色体比较定位研究 ,将有望促进脊椎动物性染色体进化的深入研究     

Localization of the mouse Mcf-2 (Dbl) protooncogene within a conserved linkage group on the mouse X chromosome

A mouse cDNA probe homologous to the human MCF2 transforming sequence has been identified and partially cloned, and is used here to localize the gene on the mouse X chromosome. The human gene has been physically mapped to within 60 kb of the gene for coagulation factor IX, within a large conserved linkage group between the mouse and human genomes which extends from HPRT to G6PD on the X chromosomes of both mammalian species. In situ hybridization of the mouse Mcf-2 probe onto mouse metaphase chromosomes indicates that this gene lies in the same region of the X chromosome as Cf-9, the mouse gene for coagulation factor IX. Moreover, segregation of species-specific genomic DNA polymorphisms for Mcf-2 and Cf-9 in a total of 203 individuals derived from two large interspecific mouse backcross populations (which are also segregating for 17 other X-linked molecular markers) demonstrates that the mouse genes are separated by only 0.5 +/- 0.5 cM. Despite this short distance we were able to order Mcf-2 and Cf-9 relative to one another and other genes in this region. The mouse gene order Hprt-Cf-9-Mcf-2-G6pd predicts a similar ordering of genes on the human X chromosome, a gene order which has only recently been demonstrated by physical mapping. Thus, the map location and linkage relationships of the Mcf-2 gene are similar in man and mouse, and this unique protooncogenic locus is part of a conserved linkage group on the mammalian X chromosome.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Genetic and physical mapping of barley telomeres

Barley (Hordeum vulgare L.) telomeres were investigated by means of pulsed field gel electrophoresis (PFGE) and in situ hybridization. In situ hybridization showed that a tandemly repeated satellite sequence has a subtelomeric location, and is present at thirteen of the fourteen chromosome ends. PFGE revealed that this satellite sequence is physically close to the telomeric repeat. Pulsed field gel electrophoresis was then used for segregation analysis and linkage mapping of several telomeric and satellite loci in a segregating doubled-haploid population. The telomeric repeat displayed a hypervariable segregation pattern with new alleles occurring in the progeny. Eight satellite and telomeric sites were mapped on an restriction fragment length polymorphism (RFLP)-map of barley, defining the ends of chromosome arms 1L, 2S, 3L, 4S, 4L, 5S and 6. One satellite locus mapped to an interstitial site on the long arm of chromosome 3. The pyhsical location of this locus was confirmed by in situ hybridization to wheat/barley addition line 3.     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

Sequences homologous to the human D1S1 locus present on human chromosome 3.

We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies.