Number Of Human Chromosomes Research Articles

The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes

A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consenus Alu sequence, were used to amplify inter- Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by bluntend ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homolog of chromosome 11. All the positive clones found were mapped to within the q23–q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes ( 7 8 ) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequence as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

Using Human Pedigrees to Teach Mendelian Genetics

Radical Genetic Findings Overturn Mendel's Laws. article bearing this headline (Raeburn 1989) reported on an address given by medical geneticist Judith Hall at a July 1989 conference at Jackson Laboratory in Bar Harbor, Maine. I was at Bar Harbor and heard Hall's challenging presentation. She discussed the phenomenon of which is germline specific modifications which produce functional differences in expression of genetic material . . . depending on whether the genetic material comes from the mother or the father (Hall 1989). Differential activity of maternally and paternally derived chromosome regions has been reported in mice (Cattanach & Kirk 1985) and more recently in humans (Knoll, et al. 1989). Among the examples of imprinting that Hall discussed is the difference in syndromes produced by the deletion of a small segment of the long arm of chromosome 15 in humans. If the deletion is inherited from the father, the resulting array of symptoms is referred to as Prader-Willi Syndrome. If the deletion is maternally inherited, it results in a different set of associated symptoms, Angelman syndrome. While such a discovery is dramatic and seems to be an exception to classical Mendelian thinking, keep in mind that, contrary to the sensational headline, Mendel's laws have not been overturned. Imprinting appears to be an exception to classical Mendelism just as linkage is an exception to independent assortment, but the more than 4,000 Mendelian genetic conditions that have been identified in humans argue for the continued viability of Mendelian genetics. McKusick (1969) states, The chief method of genetic study in man is the observation of patterns, that is, the patterns of distribution of genetic traits in kindreds. Similarly, Fuhrman and Vogel (1983) state, A detailed gives us the necessary basic information for all further genetic considerations. Despite the many technological advances of the last 20 years, pedigrees of Mendelian traits remain the most basic analytic tool of human and medical geneticists. Indeed, many of the advances in technology have enhanced the usefulness of the method, allowing one to trace, for example, the inheritance of unique DNA markers throughout a human kindred. It was an advance of this type that led to assigning the gene for Huntington's Disease to human chromosome number 4. Human pedigrees are also useful tools in the classroom, where they can serve to introduce students to the basics of Mendelian principles of inheritance. challenges afforded the student by analysis can be as rigorous and demanding as those associated with the analysis of data collected from investigations involving experimental organisms. Moreover, many beginning biology students will identify personally with the analysis of human pedigrees, where they may not have their interest piqued by the study of guinea pigs, drosophila, maize (Indian corn) or pea plants. purpose of this article is to present suggestions for using human pedigrees in the classroom and to provide a pedigree that can be used to make a transparency for overhead projection or a spirit master for preparing multiple copies for classroom distribution. generic has been prepared in a manner that can be readily modified by the instructor to provide students a host of variants to analyze.

Gene for human CD59 (likely Ly-6 homologue) is located on the short arm of chromosome 11

The CD59 (MEM-43) antigen, which probably is a human homologue of mouse Ly-6 antigens, is a broadly expressed Mr 18,000-25,000 human leucocyte surface glycoprotein recognized by monoclonal antibody MEM-43. Ten mouse-human T-lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, were analyzed for expression of CD59 by indirect immunofluorescence and immunoblotting with MEM-43 antibody. Karyotypic analysis of the tested clones showed that the presence of human chromosome 11 correlated with the expression of CD59 in all clones tested. Three other human chromosome 11-encoded antigens, 4F2 (Trop-4), Leu 7 (HNK-1, CD57), and lymphocyte homing receptor, were expressed concordantly with CD59. A more exact localization of the gene for CD59 was obtained by the study of Chinese hamster-human cell hybrids containing short or long arm deletions of human chromosome 11. CD59 segregated with hybrids containing part of the short arm of human chromosome 11, but not with the hybrids containing the long arm. Based on these studies we assign the gene for CD59 to region p14-p13 of the short arm of chromosome 11.

Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.

Chromosomal localization of the human osteocalcin gene.

The human osteocalcin gene was assigned to chromosome 1 by Southern blot analysis of DNAs from a panel of mouse-human somatic cell hybrids with limited numbers of human chromosomes and the complete complement of murine chromosomes. By Southern blot analysis of DNAs from mouse-human hybrids that retain specific segments of human chromosome 1, we have determined that the locus of the human osteocalcin gene is on the long arm of chromosome 1, distal (telomeric) to the -spectrin gene. Osteocalcin is a bone specific protein and it is note worthy that another osteoblast product, the bone/liver/placental alkaline phosphatase gene has also been mapped to chromosome 1.

The gene for human lymphocyte homing receptor is located on chromosome 11

The mouse monoclonal antibody Hermes-3 recognizes the human lymphocyte homing receptor. A panel of mouse-human T lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, was analyzed for expression of human homing receptor by indirect immunofluorescence and immunoprecipitation of radiolabeled cell lysates with Hermes-3 antibody. Karyotypic analysis of the tested clones showed that the expression of human homing receptor correlated to the presence of human chromosome 11 in all but one clone. However, concanavalin A induced a weak to moderate expression of the homing receptor in this clone, but not in a chromosome 11- clone. Another clone, heterogeneous for the expression of homing receptor, was separated into a Hermes-3+ and a Hermes-3- fraction with a fluorescence-activated cell sorter. Karyotypic analysis performed after sorting showed human chromosome 11 to segregate with the Hermes-3 antigen. To confirm these data we correlated the expression of two chromosome 11-coded antigens, Trop-4 and Leu-7, with the expression of the homing receptor. In our hybrid clones these three antigens were expressed concordantly. The gene coding for the human lymphocyte homing receptor recognized by Hermes-3 is thus assigned to chromosome 11.

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization.

Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluorescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluorescence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase in consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Predominant association of adenovirus type 12 DNA with human chromosome 1 early in productive infection

In human KB cells productively infected with human adenovirus type 12 (Ad12), viral DNA can recombine with cellular DNA. The functional significance of this covalent linkage is not understood. It is, however, conceivable that very early in the infection cycle integrated viral genes gain an advantage by being transcribed more effectively. In the present study, it has been shown that Ad 12 DNA is associated predominantly with human chromosome number 1 and one chromosome in the C II group. This association has been detected as early as 2 hr after infection by in situ hybridization. Further work will be directed toward the analysis of viral transcription products within the first 2 hr of the infection cycle.

Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells

We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

Microcell-mediated transfer of a single human chromosome complements xeroderma pigmentosum group A fibroblasts.

Chromosomes from an immortalized aneuploid human fibroblast cell line were randomly tagged with the selectable marker neo by transfection with the plasmid pSV2neo. Somatic cell fusions between transfected human cells and mouse A9 cells generated pools of G418-resistant human-mouse hybrid clones containing various numbers of human chromosomes. Microcell-mediated chromosome transfer from the hybrid pools to xeroderma pigmentosum complementation group A (XP-A) cells in culture and selection for G418-resistant colonies resulted in the identification of XP cells with enhanced resistance to ultraviolet radiation. Screening of subclones from selected pools of human-mouse hybrids facilitated the identification of hybrids containing a single neo-tagged human chromosome. Transfer of this chromosome to XP-A cells (but not to XP-F or XP-C cells) results in enhanced resistance to ultraviolet light and enhanced excision repair capacity. The identification of a single human chromosome that complements the phenotype of XP-A cells in culture provides the potential for genetic mapping of the complementing gene and for its isolation by molecular cloning.

DNA sequence and regional assignment of the human follicle-stimulating hormone beta-subunit gene to the short arm of human chromosome 11.

A human genomic DNA fragment in phage lambda containing FSHB, the gene for the beta-subunit of human follicle-stimulating hormone (FSH-beta), was analyzed and the nucleotide sequence of the region of the clone encoding FSH-beta was determined. A subclone of the lambda phage containing 67% of FSH-beta coding sequence was used as hybridization probe to determine the human chromosomal location of FSHB. A panel of mouse-human somatic cell hybrids containing reduced numbers of human chromosomes was screened with the FSHB probe; complete cosegregation of FSHB with human chromosome 11 was observed in all 26 cell hybrids tested. Analysis of a set of cell hybrids containing translocated derivatives of chromosome 11 further localized FSHB to the human chromosome region 11p11.2----11pter. A Hind III restriction fragment length polymorphism (RFLP) detected by another subclone of the lambda phage containing FSHB now provides a genetic marker for this region of the human genome.

Assignment of the gene for adrenal P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase) to human chromosome 10.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities. We identified several human P450c17 cDNA clones in a human adrenal cDNA library we constructed in lambda gt10. A short clone containing the 3'-terminal 650 bases of the full-length sequence was used to examine Southern blots of DNA from normal persons and from a panel of mouse/human somatic cell hybrid lines. The pattern of hybridization of this cDNA to normal human DNA cut with 8 restriction endonucleases suggests the human genome has two (or more) P450c17 genes. The pattern of hybridization to the somatic cell hybrid cell lines, each containing a limited, known number of human chromosomes, indicates the human adrenal P450c17 gene lies on chromosome 10. The chromosomal locations of the other P450c17 genes could not be determined.

Chromosomal localization of integrated adenovirus DNA in productively infected and in transformed mammalian cells

The in situ hybridization technique has been used to localize adenovirus type 12 (Ad12) genomes on specific chromosomes of Ad12-transformed hamster cells as well as on specific chromosomes of human cells productively infected with Ad12. Hamster cell lines T637 and A2497-2 contain 20 to 22 and 17 copies of Ad12 DNA, respectively, in an integrated form. The results of in situ hybridization experiments demonstrate that the Ad12 DNA molecules are predominantly located on chromosomes 2, 7, 11, to 13, and 15 in cell line T637, and on chromosomes 7, 20, and perhaps 16 to 19 in cell line A2497-2. These data further document that viral DNA is chromosomally integrated and does not persist in a huge extrachromosomal, episomal structure. There is evidence from previous work that adenovirus DNA can also be covalently linked to human cellular DNA after productive adenovirus infection of human cells. In keeping with these earlier findings, we have now been able to show by in situ hybridization experiments that early in productive infection, i.e., 2 or 6 hr after infection, Ad12 genomes are predominantly associated with a limited number of human chromosomes. It appears biologically significant that these chromosomes, which belong to groups A and C II, have been the same ones in several independent infection experiments. Other chromosomes may also carry smaller amounts of viral DNA. In situ hybridization of Ad12 DNA to chromosomes of uninfected human cells yields no significant chromosomal association of viral DNA. The signal to noise ratio of grain counts over chromosomes from Ad12-infected cells to areas devoid of chromosomes is 5.6. The results presented raise the question of whether Ad12 DNA can integrate at selective sites in human chromosomes and whether this insertion event plays an essential role in early steps of viral transcription or replication. There is no direct evidence to suggest a biologic function for viral DNA integration in the productive infection cycle.

Assignment of genes for human monoamine oxidases A and B to the X chromosome.

Monoclonal antibodies that immunoprecipitate human monoamine oxidase (MAO) A or human MAO B, but not the corresponding mouse enzymes, were used to assay for the presence of immunoprecipitable MAO A or MAO B (presumably coded by the respective human genes) in mouse-human hybrid somatic cell lines containing small numbers of human chromosomes. The results were as follow: Extracts of a human lymphoblastoid x mouse hepatoma hybrid line that retained the human X chromosome contained immunoprecipitable MAO B, while a similar hybrid line that contained the same human chromosomes, except for the human X, did not. Extracts of a human fibroblast x mouse neuroblastoma hybrid cell line, whose human chromosomal material consisted solely of the X, contained both immunoprecipitable MAO A and MAO B. Extracts of a related hybrid line, whose human chromosomal material consisted solely of an autonomous fragment and a fragment translocated to a mouse chromosome, contained immunoprecipitable MAO A. However, the level of immunoprecipitable MAO B activity in extracts of this hybrid was low or undetectable. Among extracts of 33 human fibroblast x mouse hepatoma hybrids that had been selected for expression of the X-linked human enzyme HPRT, 60% contained immunoprecipitable MAO B. This figure was comparable to the 58% that expressed the X-linked human isozyme for glucose-6-phosphate dehydrogenase (G6PD). When 11 of these hybrid lines, which contained immunoprecipitable MAO B and human HPRT, were selected for loss of HPRT, all lost immunoprecipitable MAO B in addition to HPRT. These data demonstrate that genes controlling the expression of MAO A and MAO B, which can be immunoprecipitated with the human-specific monoclonal antibodies, are located on the human X chromosome. Properties of the immunological epitopes recognized by the monoclonal antibodies suggest that the X-linked genes detected in this study are probably structural genes for the enzymes.

Regional assignment of the erythropoietin gene to human chromosome region 7pter→q22

The chromosomal location of the human gene for erythropoietin (EPO) was determined by Southern blot hybridization analysis of a panel of human-mouse somatic hybrid cell DNAs. DNAs from cell hybrids containing reduced numbers of human chromosomes were treated with the restriction enzyme PstI and screened with a cloned human EPO cDNA probe. EPO is assigned to human chromosome 7 based on the complete cosegregation of EPO with this chromosome in all 45 cell hybrids tested. A cell hybrid containing a translocated derivative of chromosome 7 localizes EPO to 7pter----q22. A HindIII restriction fragment length polymorphism is detected by hybridization of the EPO cDNA probe to human genomic DNA.

Genes for the ?H? subunit of human ferritin are present on a number of human chromosomes

DNA has been extracted from hamster-human and mouse-human hybrid cell lines, restricted with EcoRI, and hybridised to a probe for the H subunit of human ferritin, pDBR2. Sequences highly homologous to this probe have been found on at least eight human chromosomes: 1, 2, 3, 6p21----6cen, 11, 14, 20, and Xq23-25----Xqter. Only the gene on chromosome 11 appears to be expressed in these hybrids.

Segregation of the NK-sensitive phenotype in human × mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human × mouse interspecies somatic cell hybrids. Highly NK-sensitive (NK s) human tumor cells were fused with NK resistant (NK r) mouse fibroblasts (LMTK−) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NK s erythroleukemia cells (K-562) or NK s retinoblastoma cells (Y-79) with LMTK − displayed an intermediate NK-sensitive phenotype. One Y-79 × LMTK − hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NK r RAJI cells generated NK-resistant hybrids when fused with LMTK− fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 × LMTK −) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.

History and present status of human chromosome studies.

Until the second half of the century most advances in cytogenetics were limited to plant and invertebrate systems. Although there was great interest in mammalian and human cytogenetics, the technical difficulties encountered in making preparations prevented consistent progress. In addition to the desire to determine normal chromosome number and morphology in man and mammals, there was great interest in chromosome changes in malignancy. As early as the late 1800s pathologists observed unequal chromatin distribution in daughter tumor cells. In the early 1900s Boveri (8) developed a somatic mutation hypothesis of the origin of cancer based not on observations in cancer cells, but on his studies on sea urchin eggs. In these materials he observed unequal chromosome distributions resulting in aneuploidy and reasoned that changes such as these could play a vital role in the alteration of the cell from the normal to the malignant state. The desire to obtain the normal human karyotype that could then be compared to tumor cells was one of the main goals in studying human chromosomes. Reports from the 1700s that related soot to the scrotal cancers of chimney sweeps and the observations that had been made on induced chromosome abnormalities produced by phenols in allium meristems added to this interest (33,34). As with most discoveries the developments that permitted the determination of the human chromosome number and karyotype was not the result of a single event or breakthrough but of building on knowledge accumulated over the years by several investigators. The case of determining the human chromosome number (60) was made possible by combining four techniques. The first of these was the use of cell culture. This technique yields a higher mitotic index than most in vivo methodologies, but more important it made it possible to obtain single cell suspensions that make cells from organized tissues equivalent to cells from ascites tumors. With cells in suspension, even distribution of chemicals and solutions to all cells and manipulations of various kinds that improve the quality of the preparations are possible. The second technique was the use of colchicine to pretreat the cells. The effects of colchicine on plant chromosomes had been reported by Blakeslee and Avery (7) and also by Levan (31). By interrupting the spindle, colchicine collects mitotic figures and increases the contraction of individual chromosomes. At the time the

Localization of a gene which complements branched-chain amino acid transaminase deficiency to the short arm of human chromosome 12.

A human X Chinese hamster somatic cell hybrid (J1) containing only one human chromosome (number 11) does not express branched-chain amino acid transaminase and, therefore, cannot grow if the branched-alpha-keto acids are substituted for the branched-chain amino acids in the growth medium. J1 cells, which are glycine auxotrophs (Gly-A), were fused with normal human lymphocytes and the resulting hybrids selectively isolated in glycine-free medium. A total of 16 primary and 46 secondary clones were analyzed for isozymic and nutritional markers. Cytogenetic analysis with chromosome banding was also performed on selected hybrid clones. The results provide evidence that branched-chain amino acid transaminase is syntenic with lactic dehydrogenase B and is located on the short arm of human chromosome 12.