Analysis Of Human Chromosomes Research Articles

The relationship between sperm chromosomal abnormalities and sperm morphology in humans

It has been suggested that an assay for sperm morphology might prove useful as an initial screen in evaluating men at risk for an increased frequency of sperm chromosomal abnormalities. In this study, the technique for analysis of human sperm chromosomes after penetration of hamster eggs was employed to determine whether there is an association between the frequency of chromosomally and morphologically abnormal sperm. 30 healthy men of proven fertility were studied. The ages of the donors ranged from 22 to 55 years. The analysis was performed “blindly” so that the technician analysing the chromosome spreads had no knowledge of the age of the donors or of the individual frequencies of morphologically abnormal sperm. There was no significant relationship between the proportion of morphologically abnormal sperm and the proportion of chromosomally abnormal sperm when controlled for age. This was true for the total frequency of chromosomal abnormalities and also for numerical and structural chromosomal abnormalities. These results suggest that an assay of morphology is not a good indication of chromosomal normality in human sperm.

Diagnostic Approach to the Newborn with Ambiguous Genitalia

When assessing an infant with ambiguous genitalia, there are some important points to remember: Do not delay the evaluation of a patient with ambiguous genitalia. A delay may expose the patient unnecessarily to a life-threatening situation, such as a salt-losing crisis associated with 21-hydroxylase deficiency or one of the testosterone biosynthetic defects. Also, it is unfair to expect the family to be able to deal emotionally with the uncertainty of unresolved gender assignment. Never perform a buccal smear. There is absolutely no indication for performing a buccal smear at any point in the evaluation of any patient with ambiguous genitalia. Use of a buccal smear to determine gender assignment was helpful decades ago, before analysis of human chromosomes was possible. With current cytogenetic methodologies that can detect chromosomal mosaicism and subtle abnormalities of the X and Y chromosomes, there is never an indication to perform a buccal smear. Never make a gender assignment on the basis of the appearance of the external genitalia alone. The chromosomal sex and gonadal sex need to be determined first. The parents' feelings regarding the desired sex of the child, gender identity and fertility need to be discussed. Once there are sufficient data, gender assignment can be made. Gender assignment should be made as soon as possible after birth, but absolutely should be made by 18 months of age, when children develop gender identity. Never equivocate in the assignment of gender. The parents and the physicians must be absolutely certain of the gender assignment and must view their decision as final. Follow-up visits with the family ideally include an assessment of their acceptance of the gender assignment of the child. If there is ambivalence, steps must be taken to identify the source of ambivalence and to clarify the issues.

Sperm chromosome analysis as a new system to test human X- and Y-sperm separation.

The correlation between the percentage of F-body-positive spermatozoa (F-sperm) and that of Y-chromosome-bearing spermatozoa (Y-sperm) determined by human sperm chromosome analysis was examined after spermatozoa were separated by an albumin column method [Ericsson et al, Nature 246:421-424:1973]. The percentages of F-sperm were 43.1 +/- 4.8 (36.1-48.8) and 45.8 +/- 8.3 (35.3-66.5) before and after separation, respectively. A slight but significant (p less than 0.05) increase in F-sperm after the separation was observed in two semen donors. A total of 570 sperm karyotypes was analyzed and the percentage of Y-sperm ranged from 36.0 to 59.1 with a mean of 47.3. In one semen donor, the percentage of Y-sperm (45.6) was significantly (p less than 0.05) higher than that of F-sperm (35.3) after separation. In the remaining nine semen donors, the results obtained by chromosome analysis corresponded well with those obtained by F-body scoring. The validity of the albumin column method in isolating a Y-sperm-rich sperm population is discussed.

Detection of BrdUrd incorporation in mammalian chromosomes by a BrdUrd antibody

A bromodeoxyuridine antibody staining technique (BAT) was applied for the analysis of human chromosomes of different chromosomal band resolution. For this purpose lymphocyte cultures were synchronized and labeled with bromodeoxyuridine during the second half of the S-phase. Generally BAT was found comparable to GTG banding though some prominent GTG bands and the constitutive heterochromatin exhibit less intense staining with this technique.

Analysis of human chromosome 11 by somatic cell genetics: reexamination of derivatives of human-hamster cell line J1.

Through the fusion of a CHO cell population to a human cell population, a hybrid cell line which has lost all human chromosomes except chromosome 11 was derived. This cell line, J1, does not appear to segregate human chromosome 11 during growth. A series of deletion segregants were isolated from J1 which had lost a portion of either the long, short, or both arms of chromosome 11. This panel of deletion segregants was used for mapping a number of genetic markers on the short arm of chromosome 11. Karyotypic analysis led to the interpretation that derivatives of J1 selected for the loss of cell surface antigens encoded by genes on the short arm of the chromosome had simple terminal deletions of this chromosome arm. More recently, we have applied recombinant DNA and in situ hybridization techniques to the analysis of the structure of chromosome 11. In the course of this analysis, we have obtained data that indicate that all J1 deletion segregants retain a small chromosomal segment containing the structural genes for insulin and HRAS1. Analysis of in situ hybridization data indicates that in cell lines in which a chromosome 11 fragment cannot be identified by karyotype analysis, human DNA has been translocated to a Chinese Hamster chromosome. These results suggest that the original interpretation of the karyotypes of deletion segregants derived from J1 as simple terminal deletions is not correct. A reanalysis of gene localization studies based on these deletion segregants suggests that some assignments of genes to specific bands on chromosome 11 should be reconsidered. In particular, data on additional deletion segregants are consistent with localization of the beta-globin gene complex to band 11p15. The data presented here suggest that in several hybrid derivatives of J1, a continuous DNA segment of approximately 10(7) base pairs in length which includes the insulin and HRAS1 (cellular homolog of retroviral oncogene Harvey ras) genes has been isolated from the remainder of the human genome. We propose that the stability of chromosome 11 in the original hybrid was due to complementation of a genetic defect in the original CHO cell parent by a gene located in close physical proximity to the insulin and HRAS1 genes on chromosome 11. Data are presented which test and support this hypothesis.

Familial Hodgkin's disease a clinical and laboratory investigation

A three to sevenfold increased risk of Hodgkin's disease has been noted in families of patients. We report the first family in which all four of a four member sibship had Hodgkin's disease. In these four sibling cases, we were able to explore markers of infectious etiologic factors by measuring antibodies to Epstein-Barr virus (EBV) and human T-cell lymphotropic virus (HTLV), and possible genetic risk factors by human leukocyte antigen (HLA) typing and chromosome analysis. All three men were diagnosed within six months of their 30th birthday. HLA typing revealed that all four siblings share the DR5 antigen, and two siblings share HLA-B27. Chromosome analysis on peripheral lymphocytes revealed no abnormalities. A detailed, structured interview failed to elicit evidence of unusual exposures. The EBV antibody titers are probably within the normal range and none of the family had antibodies to HTLV-III. Observations on this family suggest that genetic factors play a greater role than environmental factors in the etiology of familial Hodgkin's disease.

Human chromosome analysis by machine

Computer assisted chromosome analysis is now becoming routine in human cytogenetics laboratories, but the initial hopes of the machine developers have not been realized. Despite the sophistication of the software programs, computers still have difficulty with simple tasks such as chromosome counting. On the other hand, they can find metaphases very rapidly and accurately, and greatly simplify karyotype production.

Analysis and sorting of human chromosomes by flow cytometry

Analysis and sorting of human chromosomes by flow cytometry

Chromosome localization of the human renin gene (REN) by in situ hybridization.

Previous studies by Southern blot analysis of human X mouse somatic cell hybrids localized the renin gene to region p21----qter of human chromosome 1. Using a DNA insert encoding exons 2-5, the renin gene was mapped to human chromosome bands 1q25----q32 by in situ hybridization. The sublocalization of the renin gene will facilitate subsequent detailed linkage analysis of human chromosome 1.

Prophase chromosome unique band sequences: definition and utilization.

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. The feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole-chromosome recognition. Through the use of prophase idiograms at the 850-band stage (Francke, 1981) and a systematic comparison system, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences, each of which has a banding pattern that is recognizable and distinct from any other nonhomologous chromosome portion. The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information.

Flow-sorting analysis of normal and abnormal human genomes.

Flow analysis of human chromosomes is a powerful new approach to study the human genome. Human chromosomes can be resolved almost completely as a result of improvements in metaphase chromosome suspension preparations, new fluorescent dye combinations, and advances in sorter optical design. We have applied this technique to map genes, detect abnormal chromosome constitutions, and construct individual recombinant chromosomal DNA libraries.

A cytochrome P-450 gene family mapped to human chromosome 19.

We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2----qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1----qter. We propose the designation P450PB for this locus.

Variable numbers of pepsinogen genes are located in the centromeric region of human chromosome 11 and determine the high-frequency electrophoretic polymorphism.

A panel of 26 mouse-human somatic cell hybrids containing different human chromosome complements was analyzed with a cloned human pepsinogen cDNA probe to determine the chromosomal location and the number of genes encoding these proteins. A complex containing variable numbers of pepsinogen genes was localized to the centromeric region of human chromosome 11 (p11----q13). Examination of somatic cell hybrids containing single copies of chromosome 11 and the corresponding human parental cell lines revealed a restriction fragment length polymorphism determined by pepsinogen haplotypes that contained two or three genes, respectively. Concurrent studies of DNA from individuals exhibiting the most common pepsinogen electrophoretic phenotypes with exon-specific probes demonstrated that the absence of one gene among the different restriction fragment patterns correlated with the absence of one specific isozymogen (Pg 5). Thus, our studies demonstrate that this genetic polymorphism involving intensity variation of individual pepsinogen isozymogens results from chromosome haplotypes that contain different numbers of genes. The regional localization of this polymorphic gene complex will facilitate detailed linkage analysis of human chromosome 11.

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.

The LLNL high-speed sorter: design features, operational characteristics, and biological utility.

This report describes a dual-beam, high-speed sorter (HiSS) with a droplet production rate of 220,000 s-1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.

Human Chromosome Analysis by Flow Cytometry

Human Chromosome Analysis by Flow Cytometry

Flow cytometry isolation and improved visualization of sorted mouse chromosomes: Purification of chromosomes X and ISO-1 from cell lines with Robertsonian translocations

While analysis and sorting of human chromosomes by flow cytometry has been widely used, isolation of a pure mouse chromosome remains very difficult, since most murine chromosomes are quite similar in size. To overcome this problem, we have analysed mouse cell lines having either Robertsonian translocations or isochromosomes. The resulting metacentric chromosomes are very different in size and in morphology from normal mouse acrocentric chromosomes. These characteristics have been analysed by computer-monitored flow cytometry, facilitated by improvements in the chromosome extraction procedure. Signals characteristic of the iso-lq chromosome in cell line PCC4azaR1, and of the normal X chromosome in the mouse strain 22CD have thus been obtained. These chromosomes have been sorted and can be easily recognized by fluorescence microscopy when collected onto serum-albumin-coated microscope slides. The technical modifications made, coupled with the existence of a great diversity of metacentric chromosomes resulting from Robertsonian translocations, should allow the purification of a number of different mouse chromosomes.

Automatic analysis of G-banded human chromosomes by fast scanning microscope photometry. I. Basic principles and computer control.

A high resolution scanning microscope photometry system has been developed for use with standard G-banded chromosome preparations. Various alternative scanning methods and their limitations are reviewed. The computer algorithms which we use are described for the manipulation of the digitized image as produced by the microscope photometer; these include noise reduction and contrast enhancement as well as altering the orientation of the chromosome.

Preparation of chromosome suspensions for flow cytometry

Certain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4 degrees C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.

Regional localization of alpha-galactosidase (GLA) to Xpter----q22, hexosaminidase B (HEXB) to 5q13----qter, and arylsulfatase B (ARSB) to 5pter----q13.

Two series of somatic cell hybrids were made by fusion of human cells with karyotypes 46,X,t(X;2;15)(q22;p12;p12) and 46,XX,t(5;7)(q13;p15) and rodent cells. Chromosome and isozyme analysis of human chromosomes and gene products in the hybrids localized GLA to Xpter----q22, HEXB to 5q13----qter, in both cases narrowing the regional assignments, and ARSB to 5pter----q13.