Interspecific Somatic Cell Hybrids Research Articles

An Entirely Novel Form of α Thalassemia in Patients from the South Pacific Linked to Chromosome 16.

Although a great deal is known about the molecular mechanism underlying α thalassemia (thal), which is characterized by reduced or absent α-globin expression, the molecular basis of α thalassemia in Pacific Islanders is not fully understood. Previously single α gene deletion alleles (−α3.7 and −α4.2) were the only types of α thalassemia identified in this population, however these genotypes do not account for several cases of Hb H disease in the Melanesian Islanders of Papua New Guinea (PNG) and Vanuatu. To date, no mutations have been found in the α globin gene cluster of these individuals. To determine the cause of α thalassemia in this population, we studied 35 individuals from three unrelated families of Pacific Islanders. 16 of them had classical Hb H disease characterized by mild to moderate anemia (Hb; 10.3 ± 2.3 g/dL), microcytosis (MCV; 66 ± 3.5 fL), hypochromia (MCH; 19.8 ± 1.6 pg), positive Hb H by electrophoresis (5–18%) and numerous Hb H inclusion bodies in peripheral blood. Gene mapping revealed that six had normal genotypes (αα/αα) while the rest were heterozygotes for α+ thalassemia (−α/αα). Preliminary sequence analyses demonstrated that the α globin genes and their regulatory elements were entirely normal casting some doubt on whether this novel form of α thal is genuinely linked to chromosome 16. Using a combination of a long range haplotype analysis by MALDI-TOF to simultaneously genotype 80 SNPs covering 350 kb of the telomeric end of chromosome 16 (16p; 13.3) and several tandem repeat markers (VNTRs and HVRs), we demonstrated that the α thal phenotype is strongly associated with a single haplotype (IIIa;PNG) found in these cases. Consistent with the Hb H patients, individuals from these families and another 15 unrelated individuals (4.7%) identified in a population survey (n = 315) who were hypochromic (MCH < 25 pg), despite having normal α genes (αα/αα) and iron status, were also found to carry the same type of chromosome 16 linked to the affected haplotype. These findings indicate that this form of α thalassemia is unequivocally linked to chromosome 16.Subsequent linkage disequilibrium analysis (LD) in these cases limited the chromosomal interval containing the mutation to within 169 kb from the 16p-telomere. Extensive genomic mapping using more than 10 restriction enzymes and 37 plasmid and PCR probes together with Pulse Field Gel Electrophoresis (PFGE), covering 400 kb of 16p, excluded small deletions, insertions, rearrangements and chromosomal truncations in the affected segment of chromosome 16. Direct genomic sequencing of collectively 40 kb of the α globin genes, all known multispecies conserved sequences (MCS-R, 1–4) demonstrated no apparent mutation. Interestingly, interspecific somatic cell hybrid analysis of the affected chromosome 16 in a mouse erythroid leukemia (MEL) background did not recapitulate the phenotype of α thalassemia. These results suggest a novel mechanism, either at the genetic or epigenetic level, underlying the reduced expression of the α globin genes in the Pacific Islanders and understanding such mechanism might provide more insights on the complexity of stage- and tissue-specific gene regulation in human erythroid environment.

Definition of the blepharophimosis, ptosis, epicanthus inversus syndrome critical region at chromosome 3q23 based on the analysis of chromosomal anomalies

Blepharophimosis syndrome (BPES) is an autosomal dominant disorder of craniofacial development, the features of which are small palpebral fissures (blepharophimosis), drooping eyelids (ptosis) and a skin fold arising from the lower eyelid (epicanthus inversus). The chromosomal localization and identity of the BPES locus is not known with certainty. In the current paper, DNA samples from three individuals with a clinical history of BPES, two with interstitial deletions (cases 1 and 2) and one with a balanced translocation (case 3) all involving chromosome 3q23, were analyzed. Allele loss studies using short tandem repeat markers in cases 1 and 2 suggested that the region between the markers D3S1292 and D3S1306 was deleted in both cases. Subsequently, the derived chromosomes resulting from the translocation in case 3 were segregated in interspecific somatic cell hybrids. Analysis of the resultant hybrids showed that D3S1615 was retained in the derived chromosome 3, whereas D3S1316 was retained in the derived chromosome 4. In neither case was the marker present in the reciprocal hybrid. These results indicate that the BPES critical region lies in the D3S1615-D3S1316 interval.

Cloning mammary cell cDNAs from 17q12–q23 using interspecific somatic cell hybrids and subtractive hybridization

We have cloned human genes that are encoded in the region 17q12–q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12–q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12–q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12–q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12–q23 region.

Analysis of Interphase Cells for the Philadelphia Translocation Using Painting Probe Made by Inter-Alu-Polymerase Chain Reaction From a Radiation Hybrid

AbstractFluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two-color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome-containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.

Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two- color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome- containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.

Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.

Identification of Yeast Artificial Chromosomes Containing the Inversion 16 p-arm Breakpoint Associated With Acute Myelomonocytic Leukemia

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.

A Sequential Staining Technique for Karyotypic Analysis of Interspecific Somatic Cell Hybrids

We describe a sequential staining technique for the karyotypic analysis of interspecific mouse × human somatic cell hybrids. Fluorescence in situ hybridization of samples, previously stained using standard trypsin/Giemsa protocols, was instrumental in the identification of human chromosomes present in hybrid lines. This procedure not only provided a simple distinction between human and mouse chromosomes, but it also allowed the visualization and monitoring of human sequences present in interspecific translocations and subchromosomal fragments.

Detection of variant Ph-positive chronic myelogenous leukemia involving chromosomes 1, 9, and 22 by fluorescence in situ hybridization

Probes made by inter- Alu-PCR from an interspecific somatic cell hybrid containing only the distal half of the 9q as its only human genomic content when used for fluorescence in situ hybridization (FISH) on the cells of a chronic myelogenous leukemia (CML) patient detected involvement of chromosome 9 in a complex translocation that was not observed by standard G-band cytogenetics.

Dual Alu polymerase chain reaction primers and conditions for isolation of human chromosome painting probes from hybrid cells

A method for rapid and efficient production of chromosome- and chromosome-region specific probes for fluorescent in situ hybridization (FISH) detectable by simple fluorescent microscopy is described. The procedure is based on simultaneous use of two inter- Alu-polymerase chain reaction (PCR) primers for extraction of highly heterogeneous human DNA from interspecific somatic cell hybrids containing the chromosome regions of interest. Probes so produced do not hybridize to centromeric sequences and simultaneously band the target chromosomes, making them useful for unambiguous identification of chromosomal elements and breakpoints associated with cancer.

Assignment of the Mouse Tartrate-Resistant Acid Phosphatase Gene ( Acp5) to Chromosome 9

Tartrate-resistant acid phosphatase is a marker enzyme for osteoclasts, the multinucleated cell responsible for bone resorption. Interspecific somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map the gene encoding tartrate-resistant acid phosphatase ( Acp5) to mouse Chromosome 9. Acp5 is therefore a member of a syntenic family of genes that map to human chromosome 19p13.1-p13.3 and mouse Chromosome 9.

Regional mapping of human DNA excision repair gene ERCC4 to chromosome 16p13.13-p13.2.

Mitomycin C (MMC)-resistant interspecific somatic cell hybrids made between human cells and the MMC-sensitive, Chinese hamster ovary (CHO) excision repair-deficient UV41 cells generally contained human chromosome 16, while other human chromosomes were randomly present. MMC-sensitive and -resistant subclones were isolated from resistant clones, and resistance generally segregated concordantly with human chromosome 16 markers. UV radiation survival analysis of subclones indicated that MMC and UV resistance were correlated. Therefore, the complementing gene, Excision Repair Cross Complementing 4 (ERCC4), was assigned to human chromosome 16. Complementation of UV41 by human cells derived from patients with xeroderma pigmentosum groups A, C, D and F excluded ERCC4 from involvement in those disease syndromes. Resistant hybrids containing only portions of chromosome 16 were identified by the lack of concordance of multiple chromosome 16 markers. When such hybrids were used as a source of probe for fluorescent in situ hybridization onto normal human metaphases, the only region of chromosome 16 identified as being consistently present was 16p13.1-p13.3. Genetic marker analysis of informative hybrids with mapped probes refined the position of ERCC4 to 16p13.13-p13.2 and allowed the following order of markers within the region to be established: pter--(PRM1, D16S215)-D16S213-D16S53-(D16S214,ERCC4) -D16S3-D16S96-cen.

Extinction of liver/bone/kidney alkaline phosphatase in osteosarcoma hybrid cells

We have constructed a series of interspecific somatic cell hybrids between the human osteoblast-like osteosarcoma, TE85, and a mouse fibrosarcoma, La-t-. In these whole-cell hybrids, we observed a 10-fold reduction of human liver/bone/kidney (L/B/K) alkaline phosphatase steady-state mRNA and alkaline phosphatase protein activity. The phenomenon of loss of tissue-specific gene expression has been termed extinction. Subclones of these hybrids were isolated, which reexpressed the alkaline phosphatase gene product. These late-passage hybrids had a reduced number of mouse fibroblast chromosomes when compared to earlier passages. This suggests that a trans-acting negative regulatory element, encoded in the fibroblast genome, regulates expression of L/B/K alkaline phosphatase. This is the first evidence that extinction plays a role in the regulation of osteoblast gene expression.

Characterization of the human gene for a newly discovered carbonic anhydrase, CA VII, and its localization to chromosome 16

Six carbonic anhydrase (CA) isozymes (CA I–VI) in mammals and other amniotes have been described. We have isolated an additional CA gene from a human genomic library and designated its putative product carbonic anhydrase VII (CA VII). The gene is approximately 10 kb long and contains seven exons and six introns found at positions identical to those determined for the previously described CA I, CA II, and CA III genes. The finding of a 17-bp GT-rich segment in a position 28 bp downstream of the poly(A) + signal and the high correspondence of the 5′ and 3′ splice sites of the six introns with consensus junction sequences are consistent with the gene being functional. The 5′ flanking regions of the CA VII gene do not contain the TATA and CAAT promoter elements usually found within 100 bp upstream of transcription initiation, but do contain a TTTAA sequence 102 nucleotides upstream of the initiation codon. The 5′ region of the gene (−243 to +551) is GC-rich and contains 80 CpG dinucleotides and four possible Sp1 (GGGCGG or CCGCCC) binding sites. Northern analysis has identified the salivary gland as a major site of expression. The derived amino acid sequence of the CA VII gene is 263 amino acids long and has 50, 56, and 49% identity with human CA I, CA II, and CA III, respectively. No differences were found at any of the 39 positions that have remained invariant in all mammalian CA isozymes sequenced to date. Based on analysis of interspecific somatic cell hybrids, the human CA VII gene, CA7, was assigned to chromosome 16, with localization to the long arm at the q21–23 region by in situ hybridization. This is in contrast to the location of the CA I, CA II, and CA III gene cluster on human chromosome 8 and that of the human CA VI gene on chromosome 1.

Isolation of a human chromosome 14-only somatic cell hybrid: Analysis using Alu and LINE-based PCR

Interspecific somatic cell hybrids containing single human chromosomes are valuable reagents for localization of cloned genes and DNA fragments to specific chromosomes, for the development of chromosome-specific libraries, and for generation of hybrid cell lines containing subchromosomal regions. A CHO somatic cell hybrid containing a single, intact human chromosome 14 (MHR14) was developed and confirmed by LINE PCR amplification gel pattern, by Alu-517 PCR product dot blot hybridization, and by cytogenetic analysis. MHR14 will serve as the chromosome source for the development of a radiation map of human chromosome 14.

Human ubiquitin-activating enzyme (E1): Compensation for heat-labile mouse E1 and its gene localization on the X chromosome

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 °C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 °C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 °C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 °C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.

Myogenin is in an evolutionarily conserved linkage group on human chromosome 1q31–q41 and unlinked to other mapped muscle regulatory factor genes

Myogenin is a member of a family of muscle-specific regulatory factors which includes MyoD1, Myf-5, and Myf-6 (also called MRF4 and herculin). Extensive regions of sequence homology in genes for these three factors suggest duplication events associated with their evolution. In the present study, the chromosomal location of the myogenin gene in humans ( MYOG), mice ( Myog), and Chinese hamsters ( MYOG) was determined using in situ hybridization to human metaphase chromosomes as well as segregation analysis among interspecific somatic cell hybrid panels and interspecific backcrossed mice. We localize the gene encoding myogenin to human chromosome 1q31–q41 within a linkage group homologous with a region on mouse chromosome 1 and Chinese hamster chromosome 5. The results verify the nonlinkage of MYOG to MYOD1, MYF5, and MYF6 genes and indicate that events associated with the duplication of MYOG with respect to MYOD1, MYF5, or MYF6 loci were not chromosome-wide.

Placental Glutathione-S-Transferase-Pi mRNA Is Abundantly Expressed in Human Skin

Four overlapping cDNA clones were isolated from a lambda gt11 human placenta cDNA library using purified human IgG antibody, from a patient with bullous pemphigoid. The sequence was homologous to human placenta glutathione-S-transferase-pi (GST-pi). Using the placenta clone, epidermal cDNA clones were isolated from a human keratinocyte library. Expression of GST-pi mRNA in human skin, cultured keratinocytes and fibroblasts, and disorders of squamous hyperplasia was demonstrated by Northern blotting and in situ hybridization. Human epidermal and placental cDNA clones hybridized to the same genomic DNA fragments. Hybridization of placental cDNA to interspecific somatic cell hybrids showed retention of chromosome 11, confirming the assignment of GST 3 to the long arm of chromosome 11 by molecular means. Anti-GST-pi antibody did not give a basement membrane zone pattern, although some normal and BP sera contained antibodies to GST-pi. Human skin expresses glutathione-S-transferase-pi, which belongs to an enzyme family important for detoxification and carcinogenesis.

Localization of the human HF.10 finger gene on a chromosome region (3p21?22) frequently deleted in human cancers

The finger motif is a tandemly repeated DNA-binding domain recently identified in the primary structure of several eukaryotic transcriptional regulatory proteins. It has been proposed that some members of the finger-gene family are implicated in both normal cell proliferation and differentiation. We isolated several human finger genes by means of hybridization with a finger motif-containing DNA probe. One of these finger genes, HF.10, is expressed at low levels in a variety of human tissues and is down-regulated during the in vitro terminal differentiation of human leukemic myeloid cell lines. By in situ hybridization experiments and analysis of interspecific somatic cell hybrids we mapped the HF.10 gene to 3p21-22, a chromosome region frequently involved in karyotypic rearrangements associated with lung and renal cancer.

Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequence polymerase chain reaction

A method was recently developed for the specific amplification of human DNA sequences from interspecific somatic cell hybrids by the polymerase chain reaction (PCR) using primers directed to Alu, a short interspersed repeat element (SINE). We now show human-specific amplification using a primer to the 3′ end of the human long interspersed repeat element L1Hs (LINE). A monochromosomal hybrid containing an intact human X chromosome yielded approximately 25 discrete products, ranging in size from 800 to 4500 bp. Combination of a single Alu primer and the L1Hs primer yielded a large number of smaller products (300–1000 bp) distinct from those observed with either primer alone. Inspection of ethidium bromidestained gels showed one Alu-Alu and three Alu-L1Hs products which were present in an intact X chromosome but absent in a hybrid containing an X chromosome deleted for the single metaphase band q28. These four fragments were isolated from the gel and used as probes on Southern blots which confirmed their localization to Xq28. These results demonstrate that primers can be constructed to a variety of interspersed repetitive sequences (IRS) and used individually or in combination for the rapid isolation of DNA fragments from defined chromosomal regions by IRS-PCR.