Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.     

Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21.

A library of yeast artificial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Double screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 +/- 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in situ hybridization, 17 mapped to chromosome 21.     

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.     

A radiation-reduced hybrid cell line containing 5 Mb/17 cM of human DNA from 9q34.

Disease gene loci for tuberous sclerosis (TSC1), idiopathic torsion dystonia (DYT1), and nail-patella syndrome (NPS1) have been mapped by genetic linkage analysis to human chromosome 9q band 34. To create a resource for physical mapping and manipulation of this region of the genome, we have created a radiation-reduced hybrid cell line containing DNA from human 9q34 as its only human component. This cell line, E6B, has been characterized by Southern blot and PCR analysis using a panel of 9q markers and fluorescent in situ hybridization. We estimate that it contains 5 Mb of human DNA, equal to 17 cM of genetic distance, extending from AK1 to ABO on 9q34.     

Clinical and molecular characterization of a combined 17p13.3 microdeletion with partial monosomy 21q21.3 in a 26-year-old man

We led a clinical and molecular characterization of a patient with mild mental delay and dysmorphic features initially referred for cytogenetic exploration of an azoospermia. We employed FISH and array CGH techniques for a better definition and refinement of a double chromosome aberration associating a 17p microdeletion with partial monosomy 21q due to 1:3 meiotic segregation of a maternal reciprocal translocation t(17;21)(p13.3;q21.2) revealed after banding analysis. Brain MRI depicted partial callosal and mild diffuse cerebral atrophies, but without expected signs of lissencephaly. The patient's karyotype formula was: 45,XY,der(17)t(17;21)(p13.3;q21.2)mat,-21. FISH study confirmed these rearrangements and array CGH analysis estimated the loss sizes to at least 635 kb on chromosome 17 and to 15.6 Mb on chromosome 21. The absence of lissencephaly and major brain malformations often associated with 17p terminal deletions could be attributed to the retention of PAFAH1B1, YWHAE and CRK genes. Dysmorphic features, moderate mental impairment and minor brain malformations could result from the 21q monosomy and particularly the partial deletion of the APP-SOD1 region. Azoospermia should result from gamete apoptosis induced by a control mechanism triggered in response to chromosome imbalances. Our study provides an additional case for better understanding and delineating both 17p and 21q deletions.     

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.     

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.     

Yeast artificial chromosome mapping of the cystinosis locus on chromosome 17p by fluorescence in situ hybridization

The gene locus for cystinosis has been mapped between markers D17S1583 and D17S1584 on the short arm of chromosome 17. Using markers encompassing the cystinosis region, we assigned different yeast artificial chromosome (YAC) clones previously identified by sequence tagged site (STS) screening to 17p13.3. Three of the clones hybridized to the target 17p gene region; one of these was chimeric, hybridizing both to chromosomes 3p and 5q; two of the YACs did not contain sequences of 17p13.3. Our physical mapping has identified candidate YACs as a first step towards a positional cloning approach. Received: 28 February 1996 / Revised: 3 May 1996     

Severe psychomotor retardation in a boy with a small supernumerary marker chromosome 19p

We describe the clinical case of a nine-year-old boy with psychomotor retardation and a small supernumerary marker chromosome (sSMC) present in mosaic form. Fluorescence in situ hybridization (FISH) using centromere cross-hybridizing probes D1/5/19Z (pZ5.1), the whole chromosome paint probe 19, pool YACs19p (839B1, 872G3, 728C8), and pool YACs19q (767C4, 761C1, 786G6) demonstrated that the sSMC was derived from chromosome 19p. Based on GTG-banding and FISH analyses, the patient's karyotype was interpreted as: 47,XY,+mar.ish der(19) (:p13.3-->p11:)(839B1+, 872G3+,728C8+, D1/5/19Z+) de novo[52]/46,XY[48]. To our knowledge, only two other similar cases have been reported. This case helps to better delineate karyotype-phenotype correlations between sSMC 19p and associated clinical phenomena.     

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Étude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5–10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics. YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution. We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs. Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.     

Boy with seizures (West syndrome) and distal 7q duplication syndrome due to an unbalanced 7q;9p translocation

We report a 15 month old boy with prominent metopic suture, epicanthal folds, strabismus, low-set ears, microretrognathia, large anterior fontanel, bilateral simian creases, muscular hypotonia, and severe psychomotor retardation. He also had West syndrome. An electroencephalogram showed hypsarrythmia, and cranial MR indicated a myelinisation delay. Standard karyotyping showed additional material on one chromosome 9p. Using FISH, a terminal 7q duplication spanning 26 Mb in size and a terminal 9p deletion sized (at least) 9.1 Mb were identified. The father had a karyotype of t(7;9)(q33;p23) and the mother's karyotype was normal. The boy presented typical facial features of the distal 7q duplication syndrome but no genital anomalies attributable to his distal 9p deletion. We assume that the severe epilepsy is likely due to the trisomy 7q.     

Localization of subtelomeric sequences of human chromosomes 1q, 11p, 13q, and 16q in the higher primates

Relative phylogenetic divergence of the members of the Pongidae family has been based on genetic evidence. The recent isolation of subtelomeric probes specific for human (HSA) chromosomes 1q, 11p, 13q, and 16q has prompted us to cross hybridize these to the chromosomes of the chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo pygmaeus, PPY) to search for their equivalent locations in the great apes. Hybridization signals to the 1q subtelomeric DNA sequence probe were observed at the termini of human (HSA) 1q, PTR 1q, GGO 1q, PPY 1q, while the fluorescent signals to the 11p subtelomeric DNA sequence probe were observed at the termini of HSA 11p, PTR 9p, GGO 9p, and PPY 8p. Fluorescent signals to the 13q subtelomeric DNA sequence probe were observed at the termini of HSA 13q, PTR 14q, GGO 14q, and PPY 14q, and positive signals to the 16p subtelomeric DNA sequence probe were observed at the termini of HSA 16q, PTR 18q, GGO 17q, and PPY 19q. These findings apparently suggest sequence homology of these DNA families in the ape chromosomes. Obviously, analogous subtelomeric sequences exist in apes' chromosomes that apparently have been conserved through the course of differentiation of the hominoid species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of DNA fragments from a human chromosomal subregion by Alu PCR differential hybridization

The recent advent of Alu element-mediated PCR (Alu PCR) allows the rapid isolation of human-specific fragments from mixed DNA sources. This technique greatly facilitates the isolation of DNA fragments from specific regions of the human genome. We report a novel technique utilizing Alu PCR products as differential hybridization probes to isolate human DNA fragments from a chromosomal subregion. We used the Alu PCR products from a pair of somatic cell hybrids in which the human DNA content differs only in the 5q11.2-q13.3 region as differential hybridization probes. One hybrid (GM10114) retains an intact chromosome 5, while the other (HHW1064) contains a chromosome 5 deleted for the q11.2-q13.3 region. Phage from a flow-sorted chromosome 5 library were hybridized with the Alu PCR synthesis product from the chromosome 5 hybrid. Positively hybridizing phage were then screened with the Alu PCR product from the deletion 5 hybrid. Phage that hybridized to the Alu PCR product of the chromosome 5 hybrid but did not hybridize to the Alu PCR product of the deletion 5 hybrid were further characterized. We isolated five phage from 5q11.2-q13.3 using this differential hybridization procedure. Only one of these phage corresponded to a detectable difference between the ethidium bromide-stained Alu PCR products of the two somatic cell hybrids. This technique should be applicable to any somatic cell hybrid-deletion hybrid pair.     

Cloning of STK13, a Third Human Protein Kinase Related toDrosophilaAurora and Budding Yeast Ipl1 That Maps on Chromosome 19q13.3–ter

This report describes the identification of a cDNA encoding STK13, a third human protein kinase related to theDrosophilaAurora and the budding yeast Ipl1 kinases. After screening of a human placental cDNA library with aXenopus laeviscDNA encoding the pEg2 protein kinase and 5′ RACE on testis mRNA, a full-length cDNA was isolated. The chromosomal localization of STK13 on 19q13.3–ter between the markers D19S210 and D19S218 was established by a combination of somatic cell and radiation hybrid panel PCR screening. The localization of STK13 on human chromosome 19 was confirmed by fluorescencein situhybridization (FISH) using a genomic clone containing STK13 as a probe.     

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of familial partial 10p trisomy by chromosomal microdissection,FISH, and microsatellite dosage analysis

Unbalanced translocations are a frequent cause of multiple congenital anomalies in children. Translocations as small as 2–5 Mb of DNA are detectable by G-banding under optimal conditions. Some of these small translocations are visible but cannot be characterized cytogenetically due to the lack of characteristic banding on Giemsa preparations. We have combined chromosomal microdissection and fluorescence in situ hybridization (FISH) to identify the origin of a small translocated segment in three members of a family with a derivative chromosome 9 and multiple anomalies, including several ophthalmologic anomalies. We microdissected the abnormal region of the derivative 9 chromosome and used this DNA to generate a FISH probe. This probe hybridized to distal 10p on the metaphase spread of the proband, indicating the origin of the translocated segment. A whole 10p FISH probe confirmed the origin by hybridizing to the translocated segment of the derivative chromosome. FISH was then performed with a whole chromosome 9 painting probe and excluded the presence of a reciprocal, balancing translocation. We then studied the chromosome 10 partial duplication with microsatellite markers to better characterize the chromosomal segment that caused these phenotypic features. By examining the involved areas with distal 10p and 9p microsatellite markers, we were able to demonstrate a minimum of 9 Mb of trisomic 10p DNA with a chromosomal breakpoint between 10p14– 10p15. We then compared this family’s clinical findings to those of individuals with partial 10p trisomy who had been reported in the literature. The clinical phenotypes seen in this family are similar to, but milder than, the phenotypes of persons with the larger partial trisomies of 10p that were diagnosable by cytogenetic analysis alone. This study shows that microdissection and DNA markers can be used to precisely define small translocations that are difficult to identify by conventional G-banded chromosome analysis. Received: 29 February 1996 / Revised: 19 April 1996     

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.     

TAR-cloning of the short arm of human chromosome 7 in yeast and search for terminal sequences

A partial clone library of the short arm of human chromosome 7 was created in yeast artificial chromosomes (YAC) using TAR-cloning. The DNA of monochromosome somatic hybrid cells (mouse/human) RuRag 14-4-7-44 containing short arm human chromosome 7 was used for cloning. The clone library was screened for YACs with the human DNA; the mitotic stability of these YACs, the sizes of cloned fragments, and an independent clonal distribution in the chromosome were determined. Human YACs were tested for the presence of chromosome 7p telomeric sequences.