Intact Chromosomes Research Articles

Chapter 6 Cell Cycle Synchronization, Chromosome Isolation, and Flow-Sorting in Plants

Publisher Summary This chapter discusses the cell-cycle synchronization, chromosome isolation, and flow sorting in plants. Plant cell populations synchronously traversing the cell division cycle or accumulated within a specific phase of the cycle represent a valuable model for studying a wide range of biological questions. Techniques for cell synchronization, chromosome sorting, and the production of chromosome-specific libraries were initially developed in mammalian species. The delay in developing equivalent techniques for the flow cytometric analysis and sorting of plant chromosomes was mainly due to difficulties in obtaining chromosome suspensions that contained intact and well-dispersed chromosomes and that were free of contaminating cellular debris. The procedure for chromosome isolation involves four fundamental steps: (1) induction of cell-cycle synchrony, (2) accumulation of cells in metaphase, (3) isolation of chromosomes from the cells, and (4) flow sorting of chromosomes. For the isolation of chromosome suspensions, problems posed by the use of plant tissues include (1) difficulties in obtaining a high level of mitotic synchrony, (2) the variable degree of chromosome stickiness and clumping observed after treatment with mitotic drugs, (3) a tendency for metaphase-blocked chromosomes to split into single chromatids, (4) the presence of the rigid plant cell wall which, in comparison to mammalian cells, increases debris and inhibits the release of intact chromosomes during homogenization.

9] Microcell fusion

Publisher Summary This chapter focuses on microcell fusion. The first documented fusions of somatic cells in culture in the early 1960s resulted in the production of viable hybrid cells and produced a technology that would revolutionize genetic analysis in vitro. Parasexual approaches have been instrumental in the physical mapping of the human genome and in the identification and mapping of genes involved in growth regulation, differentiation, and neoplasia. Microcell fusion is a method of physical mapping which involves the transfer of single or small clusters of intact chromosomes from one cell to another. It is perhaps the most attractive technique for interspecific and intraspecific gene mapping because it affords a way to introduce a single chromosome, rather than the entire genome, from a donor cell into a recipient cell background. The transferred chromosome can be stably retained in the recipient cell background under dominant selective pressure. This type of monochromosomal hybrid has several advantages over whole cell hybrid clones. Microcell fusion is accomplished in five essential steps that include: (1) micronucleation of donor cells, (2) enucleation of the micronucleate cell population, (3) isolation of microcells, (4) fusion of microcells to recipient cells, and (5) selection of viable microcell hybrid clones.

The substructural organization of the chromosome core (scaffold) in meiotic chromosomes of Trilophidia annulata

SummaryThe substructural organization of chromosome cores or nonhistone proteins was studied within intact metaphase chromosomes at the second meiotic division in the grasshopper Trilophidia annulata by silver staining as well as light microscopy and whole mount electron microscopy of squash chromosomes. Our results revealed that the metaphase II chromosome contains a longitudinal, helical coiling core structure. Probably the two last organizational levels of the core packaging are achieved by helical coiling. The core structure retains the morphological characteristics of the original metaphase chromosome, surrounded by a halo of dispersed materials, which may be composed mainly of nonhistone proteins. The kinetochore is found to be connected with the chromosome core. The present findings combined with our previous observations on the helical structure of metaphase II chromosomes suggest that the folding path of the internal core structure in metaphase chromosomes is consistent with the final helical arrangement of the chromosome itself. These observations also imply that in condensed metaphase chromosomes nonhistone protein may form a compact network structure with helical appearance, which extends throughout the entire chromosome.

Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection.

Human oocytes that failed to display signs of fertilization by 44 h after intracytoplasmic sperm injection (ICSI) were processed for electron microscopic analysis. All oocytes were arrested at metaphase II. The first polar body contained intact cortical granules and chromosome clumps, which were not surrounded by a nuclear envelope but still associated with microtubules. When a second globular body was present, it always showed the same ultrastructure, indicating that it had originated from fragmentation of the first polar body and not from the resumption of the second meiotic division. The most prominent organelles of the oocyte cytoplasm were the smooth endoplasmic reticulum and mitochondria. In the oocyte cortex, cortical granules were intact, with no signs of incipient or incomplete cortical reaction. Oocyte chromosomes were found in the oocyte periphery near the locality of the first polar body extrusion. They consisted of dense aggregates of chromatin associated with microtubules. The chromatin of the injected spermatozoon was demembranated and partially decondensed. In some cases, vesicular and tubular structures, apparently of oocyte origin, were associated with the periphery of the sperm chromatin mass but they never formed a continuous layer. These data suggest that fertilization failure after ICSI is basically a failure of oocyte activation.

A variety of genetic mechanisms are associated with the Prader-Willi syndrome.

An extensive set of chromosome 15 DNA polymorphisms and densitometric analysis with four markers mapping to the Prader-Willi chromosome region (PWCR) of chromosome 15 have been used to characterize a cohort of 30 subjects with classical Prader-Willi syndrome (PWS). Molecular analysis enabled the classification of the PWS subjects into four groups: (A) 18 subjects (60%) had deletions of paternal 15q11-13 involving a common set of DNA markers. Two subjects had differently sized deletions, one larger and one smaller than the other cases. (B) Eight (27%) had maternal uniparental disomy for chromosome 15. (C) One (3%) had a marker chromosome carrying an extra copy of the PWCR. The marker chromosome was demonstrated to be of paternal origin and the two intact chromosomes were maternally derived. This case represents an apparent exception to the generally held view that PWS is associated with an absence of paternally inherited gene(s) located in the PWCR. (D) The remaining three cases (10%) had none of the above abnormalities. This last subgroup of patients has not previously been well characterized but could represent limited deletions not detectable with the markers used or abnormalities in the imprinting process. These cases represent potentially valuable resources to elucidate more precisely the fundamental disorders responsible for PWS.

Integrated Mapping Analysis of the Werner Syndrome Region of Chromosome 8

The Werner syndrome locus (WRN) is located at 8p11-p12. To facilitate eventual cloning of the WRN gene, a 10,000-rad radiation-reduced hybrid (RH) cell panel was generated to map genetic markers, sequence-tagged sites (STSs), and genes in this region. A hamster cell line carrying an intact human chromosome 8 was fused with another hamster cell line. Two sets of hybrid cell panels from 2 separate fusions were generated; each panel consisted of 50 independent clones; 33 and 34 cell lines from the 2 fusions retained human chromsome material as determined by inter-Alu PCR. The combined panel was genotyped for 52 markers spanning the entire chromosome, including 10 genes, 29 anonymous polymorphic loci, and 13 STSs. Seventeen of these markers have not been previously described. Markers near the centromere were retained at a higher frequency than more distal markers. Fluorescence in situ hybridization was also used to localize and order a subset of the markers. A RH map of the WRN region was constructed using a maximum likelihood method, giving the following most likely order: D8S131-D8S339 (GSR)-D8S124-D8S278-D8S259-(D8S71)-D8S283- D8S87-D8S105-D8S135 (FGFR1)-D8S135PB-D8S255-ANK1. A genetic map of 15 short tandem repeat polymorphic loci in the WRN region was also constructed. The marker orders from the genetic and RH maps were consistent. In addition, an integrated map of 24 loci in the WRN region was generated using information from both genetic and RH mapping methods. A 1000:1 framework map for 6 loci (LPL-D8S136-D8S137-D8S87-FGFR1-ANK1) was determined by genetic mapping, and the resulting locus order was fixed during analysis of the RH genotype data. The resulting integrated map contained more markers than could confidently be ordered by either genetic or RH mapping alone.

Characterization of chimpanzee-hamster hybrids by chromosome painting.

A panel of chimpanzee-human somatic cell hybrids was characterized by dual Alu-PCR of the chimpanzee DNA in the hybrid and subsequent hybridization of the labeled PCR products to human and chimpanzee chromosomes. In addition to the identification of the intact chimpanzee chromosomes retained in each hybrid, chromosome fragments were identified that will be useful in regional mapping. This technique also revealed the presence of centric inversions.

Recombination of Solanum brevidens chromosomes in the second backcross generation from a somatic hybrid with S. tuberosum

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC2 progeny from six fertile BC1 plants. The BC1 plants were derived from a single S. brevidens + S. tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC2, suggesting that recombination had occurred. For 8 of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n=48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.

Induction of senescence and control of tumorigenicity in BK virus transformed mouse cells by human chromosome 6.

Viral transformation models may be useful to detect and map human tumor suppressor genes. BK virus (BKV), a human papovavirus, readily transforms rodent cells but is unable to transform human cells, suggesting that oncosuppressive functions expressed in human cells control BKV oncogenic activity. We have transferred human chromosome 6 to BKV-transformed mouse pRPcT1ss1 cells. The great majority of the colonies growing in selective medium degenerated by senescence. Only five hybrid pRPcT1ss1/H6 clones maintained the immortalized phenotype of the recipient cell line. All the immortalized clones had two common regions of deletion involving bands 6q21-22 and the SOD2 gene in 6q25. Senescent colonies carried an intact chromosome 6. A specific human sequence in 6q21-22 was amplified by PCR in senescent cells, suggesting that this region harbors a gene inducing senescence. The SOD2 deletion confirms recent data on the role of the Mn-dependent superoxide dismutase in inhibition of proliferation. The monochromosomic hybrids bearing a deleted chromosome 6 showed a reverted phenotype in vitro and a significantly longer latency period before they were tumorigenic in nude mice, indicating the presence of a tumor suppressor gene in the residual regions of chromosome 6. Molecular mapping suggests that this gene is located in 6q27. The BKV transformation model detects genes inducing senescence and tumor suppressor genes on human chromosome 6 and may represent a useful system to isolate and clone such genes.

Interstitial Insertion of Retinoic Acid Receptor-α Gene in Acute Promyelocytic Leukemia With Normal Chromosomes 15 and 17

The translocation t(15;17)(q22;q21) is seen exclusively in patients with acute promyelocytic leukemia (APL) and in the promyelocytic blast crisis of chronic myeloid leukemia (CML). This translocation juxta-poses the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARA) gene on chromosome 17, resulting in the formation of a chimeric mRNA transcript. We describe a patient with the microgranular variant form of APL, with no detectable cytogenetic abnormality of either chromosomes 15 or 17, who nevertheless had juxtaposition of PML and RARA genes and expressed a chimeric transcript. Conventional cytogenetics showed the karyotype 46,XY,d-er(3)t(3;8)(p25;q12). Fluorescent in situ hybridization (FISH) with paints for chromosomes 8, 15, and 17 confirmed the presence of structurally intact chromosomes 15 and 17 and trisomy for chromosome 8q. Nevertheless, FISH using cosmid probes for PML and RARA showed their juxtaposition on one chromosome 15 homolog. Both genes were also present on their normal homologs; in addition, part of the RARA gene was still present on the remaining chromosome 17. DNA analysis by Southern blotting, performed with a variety of probes including PML, RARA and retinoic acid receptor-beta (RARB), showed a rearrangement in PML. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the existence of hybrid transcripts of 276, 455 bp and 623 bp, from PML-RARA on the der(15) chromosome, consistent with alternate exon splicing of the long form of the transcript occurring in 50% to 60% of patients with APL. Our results show that APL patients with cytogenetically normal chromosomes 15 and 17 may, nevertheless, have involvement of both PML and RARA genes defining a subgroup of APL, t(15;17)-negative/PML-RARA-positive which is analogous to Philadelphia chromosome-negative/BCR-ABL-positive CML. In this case, the presence of chimeric transcripts suggests that treatment with all-trans RA may be warranted in APL, even in the absence of detectable cytogenetic change, showing the usefulness of RT-PCR or FISH to aid diagnosis.

In vitro growth suppression and morphological change in a human renal cell carcinoma cell line by the introduction of normal chromosome 3 via microcell fusion

Cytogenetic and molecular studies of human renal cell carcinoma (RCC) have suggested that the genetic and functional losses of one or more putative tumor suppressor genes on the short arm of chromosome 3 play a crucial role in the development of this disease. To examine whether the introduction of chromosome 3 has any effects on the biology of RCC cells, we introduced either chromosome 3, 7, or 11 from normal human fibroblasts into a newly established human RCC cell line with loss of heterozygosity for 3p, via microcell-mediated chromosome transfer. Microcell hybrids containing an introduced, intact chromosome 3 showed a significant reduction in in vitro growth rate and saturation density together with morphological alteration; these properties were not altered in microcell hybrids containing an introduced chromosome 7 or 11. During long-term cultivation, one of the clones that had lost the introduced chromosome 3 showed growth properties and morphology similar to those of the parental cell lines. Thus, our findings provide additional evidence for the presence of a putative tumor suppressor gene or genes on normal chromosome 3p and indicate that the gene is a dominant, negative growth regulator whose loss promotes progressive features of the neoplastic phenotype.

Mapping the human growth hormone-releasing hormone receptor (GHRHR) gene to the short arm of chromosome 7 (7p13-p21) near the epidermal growth factor receptor (EGFR) gene.

In this report, the authors have assigned the human GHRHR gene to chromosome 7p13-p21, using polymerase chain reaction (PCR) amplification of DNA from well-defined human-rodent somatic cell hybrids. The GHRHR gene was assigned to human chromosome 7 by discordancy analysis (data not shown) of PCR amplification products from NIGMS mapping panel Nos. 1 and 2 DNA templates. The PCR primers (p[sub f], 5[prime]-GCTGCCTCATCACGCCACTGGAGTCCAC-3[prime]; and P[sub r], 5[prime]-CAGGTTTATTGGCTCCTCTGAGCCTTGG-3[prime]) amplified a 276-bp-long fragment from the 3[prime] untranslated region of the human GHRHR gene. Subsequently, they determined the location of the GHRHR gene within human chromosome 7 by PCR amplification of genomic DNA template from somatic cell hybrids that contain deletions of this chromosome. Amplification of the 276-bp DNA fragment was seen only in the cell lines that contained an intact chromosome 7 short arm. The lack of amplification using genomic DNA from 0044 Rag 1-15 and It A9 2-21-14 maps this gene to 7p13-p21. Additionally, the appropriate amplified product was observed from the human chromosome 4 containing NIGMS panel 2 cell line GM10115. This line was reported to have retained a small region of human chromosome 7 containing the epidermal growth factor receptor (EGFR) gene that is mapped to 7p12-p13. The authorsmore » conclude that the human GHRHR gene maps to the small arm of chromosome 7 within 7p13-p21 and close to the EGFR gene. This assignment is consistent with the syntenic relationship between mouse chromosome 6 and human chromosome 7 in this region.« less

Somatic cell hybrid panel and NotI linking clones for physical mapping of human chromosome 3.

To identify by positional cloning the putative tumor-suppressor genes on the short arm of human chromosome 3 that are involved in the initiation or progression of several human malignancies, we have developed human mouse somatic cell hybrids and NotI linking libraries. The somatic cell hybrids contain either the intact human chromosome 3 or its derivatives as the only human genetic material in rodent background. The somatic cell hybrid panel defines five chromosomal regions on the short arm and two chromosomal regions on the long arm of chromosome 3. Two hundred sixty-one NotI linking probes from three independently constructed linking libraries were assigned to the seven chromosomal regions. The somatic cell hybrid panel and the regionally localized NotI linking probes should facilitate the construction of genetic linkage and physical maps to identify various tumor-suppressor and disease-related genes not only on the chromosome 3p, but on the entire chromosome.

Isolation and mapping of cosmid markers on human chromosome 22, including one within the submicroscopically deleted region of DiGeorge syndrome

A genomic cosmid library was constructed from a Chinese hamster/human hybrid cell containing human intact chromosome 22 as its only human component. Of 1000 cosmids with inserts derived from human chromosome 22, 191 were tested for restriction fragment length polymorphisms (RFLPs). As a result, 64 clones detected RFLPs, including five variable number of tandem repeats systems. Of the remaining 127 cosmids, 111 detected a single copy sequence on human chromosome 22. Five somatic cell hybrids allowed us to assign all of the 64 polymorphic cosmids and 44 non-polymorphic cosmids to four different regions of human chromosome 22. In two patients with DiGeorge syndrome, one of the cosmids that had been sublocalized to 22pter-q11 detected hemizygosity. These 108 cosmid markers regionally assigned to human chromosome 22 should be useful for the construction of long-range physical maps and the identification of genetic alterations on the chromosome.

A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1-->q11.1.

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with 60Co gamma-rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22-->qter or del(8)q23--> qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybrids, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1--> q11.1.

Image analysis of fish stained specimens: A new diagnostic tool in genetics and oncology

In situ hybridization allows the detection of specific nucleic acid sequences in morphologically intact cells and chromosomes. Presently, fluorescence in situ hybridization (FISH) has reached a high detection sensitivity (defined as the smallest DNA target detectable, high DNA resolution (defined as the smallest distance in kilobasepairs between two DNA targets that can be resolved microscopically and a high multiplicity (defined as the number of different probes that can be identified simultaneously. The current status of FISH methodology:*several direct and indirect nucleic acid modifications*sensitivity: unique DNA sequences 1-5 kb*DNA resolution: metaphase ~ > 1-3 Mbp (light microscopy) interphase ~50 kbp DNA halos ~ 1 kbp*multiplicity: at least 12Progress in in situ hybridization and related technology has caused molecular cytogenetics to become an established. In particular the ability of ISH techniques to detect chromosomes and/or chromosome parts in interphase nuclei (interphase cytogenetics) has significantly contributed to this acceptance of ISH, since this technique provides statistically reliable means to study the genetic composition of cells that can not, or only by complicated techniques, be brought in mitosis.

Analysis of Drosophila chromosome 4 using pulsed field gel electrophoresis.

Previous estimates of the size of Drosophila melanogaster chromosome 4 have indicated that it is 1% to 4% of the genome or approximately 6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules of D. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from the cubitus interruptus and zfh-2 genes, which are located on chromosome 4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome 4. In DNA obtained from embryos of various D. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. The D. melanogaster chromosome 4 probes also identified a single, 2.4 Mb band in embryonic DNA from Drosophila simulans. We conclude that D. simulans chromosome 4 is substantially smaller than that of D. melanogaster, presumably owing to differences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome 4 DNA and will facilitate the molecular mapping of this chromosome.

Loss of a yeast telomere: Arrest, recovery, and chromosome loss

Yeast strains were constructed in which a single telomere could be eliminated from the end of a dispensable chromosome. In wild-type cells, elimination of a telomere caused a RAD9-mediated cell cycle arrest, indicating that telomeres help cells to distinguish intact chromosomes from damaged DNA. However, many cells recovered from the arrest without repairing the damaged chromosome, replicating and segregating it for as many as ten cell divisions prior to its eventual loss. Telomere elimination caused a dramatic increase in loss of the chromosome in all strains examined, demonstrating that yeast telomeres are also essential for maintaining chromosome stability. Thus, in spite of checkpoint and DNA damage repair systems, many chromosomes that lose a telomere are themselves destined for loss.

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.

Restoration of the cholesterol metabolism in 3T3 cell lines derived from the sphingomyelinosis mouse (spm/spm) by transfer of a human chromosome 18.

We searched for a human chromosome that would restore the cholesterol metabolism in 3T3 cell lines (SPM-3T3) derived from homozygous sphingomyelinosis mice (spm/spm). Mouse A9 cells containing a single copy of pSV2neo-tagged chromosomes 9, 11, or 18 derived from normal human fibroblasts served as donor cells for transfer of human chromosomes. Purified A9 microcells were fused with SPM-3T3 cells, and the microcell hybrids were selected in medium containing G418 antibiotics. The microcell hybrids that contained human chromosomes 9, 11, or 18 in a majority of cells were examined. The accumulation of intracellular cholesterol in the microcell hybrids containing a chromosome 18 decreased markedly, whereas in the microcell hybrids containing either chromosomes 9 or 11 it was similar to that in SPM-3T3 cells. The SPM-3T3 cells with an intact chromosome 18 were further passaged and subcloned. Clones which again accumulated intracellular cholesterol had concurrently lost the introduced chromosome 18. The abnormal accumulation was associated with a decrement in the esterification of exogenous cholesterol. These findings suggest that the gene responsible for the abnormal cholesterol metabolism in the spm/spm mice can be restored by a human chromosome 18. The gene was tentatively mapped on 18pter-->18p11.3 or 18q21.3-->qter that was lost during subcloning, thereby resulting in reaccumulation of the intracellular cholesterol.