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PubMed2001

High-resolution BAC-based map of the central portion of mouse chromosome 5.

The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable.

Animals↗
PubMed1996

Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities.

A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach. We used an alternative approach of genomic walking, starting from a subtelomeric sequence, Tel-Bam3.4. present on a number of human chromosomes including 22q. This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q. The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q. The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22. In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis. Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations.

Cerebroside-Sulfatase↗
PubMed1993

Molecular dissection of an extrachromosomal amplicon reveals a circular structure consisting of an imperfect inverted duplication.

A mouse fibroblast line, B-1/50, with a 4300-fold amplification of the adenosine deaminase gene locus (Yeung et al., 1983, J. Biol. Chem. 258: 8338-8345), was shown by in situ hybridization to harbor the amplified sequences on variously sized extrachromosomal elements. We show here that the smallest circle is approximately 500 kb. We describe a facile screening technique for identifying cosmid and yeast artificial chromosome (YAC) clones derived from the amplicon. A closed molecular map was generated by arranging the cosmids and YACs into a contig spanning over 250 kb of the adenosine deaminase gene locus. YACs from the two ends of this contig were shown to delimit a 250-kb inverted duplication. Long-range mapping of a SalI partial digest of B-1/50 DNA is also consistent with the interpretation that the 500-kb adenosine deaminase amplicon in B-1/50 cells is an inverted duplication. The finding that this amplicon is the only or predominant structure containing amplified sequences in the B-1/50 cell line suggests that such structures are not inherently prone to high frequency rearrangement, even when present at such high copy number. This study provides the first molecular description of the structure of an episome involved in mammalian gene amplification. The implications of this finding for models of gene amplification and episome formation are discussed.

Adenosine Deaminase↗
PubMed1999

The application of AFLP fingerprinting to construct a YAC contig containing ADH2 and MTP on sheep chromosome 6.

The low density of genetic markers on livestock maps limits progress in positional cloning projects. We demonstrate a strategy of combining comparative mapping with AFLP fingerprinting to develop physical maps in a defined region of the sheep genome. Sequence tagged sites for alcohol dehydrogenase 2 (ADH2) and microsomal triglyceride transfer protein (MTP) were developed and used to screen a sheep yeast artificial chromosome (YAC) library. Nine YACs were identified containing the microsatellite marker BM1329 and either ADH2 or MTP. Additional markers in the region were not available, and AFLP analysis was developed to identify sheep-specific bands within the YACs to determine their degree of overlap. Fourteen bands common to more than one YAC were analysed and provided the markers necessary to develop a YAC contig containing the three STS markers. One YAC (yac260B5) containing all three markers (ADH2, MTP, and BM1329) was mapped to sheep chromosome 6q1.6-->q1.8 by FISH analysis.

Alcohol Dehydrogenase↗
PubMed1996

Construction and characterization of three region-specific microdissection libraries for human chromosome 18.

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and Mbol linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50-600 bp, with a mean of 180-220 bp for the three libraries. The libraries contained approximately 40-60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human genomic HindIII fragments hybridized to each microclone were determined and microclones cross-hybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating highly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.

Chromosome Mapping↗
PubMed2004

Sequencing the maize genome.

Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Multiple pathogenic and benign genomic rearrangements occur at a 35 kb duplication involving the NEMO and LAGE2 genes.

The X-linked dominant and male-lethal disorder incontinentia pigmenti (IP) is caused by mutations in a gene called NEMO (IKK-gamma). We recently reported the structure of NEMO and demonstrated that most IP patients carry an identical deletion that arises due to misalignment between repeats. Affected male abortuses with the IP deletion had provided clues that a second, incomplete copy of NEMO was present in the genome. We have now identified clones containing this truncated copy (Delta NEMO) and incorporated them into a previously constructed physical contig in distal Xq28. Delta NEMO maps 22 kb distal to NEMO and only contains exons 3-10, confirming our proposed model. A sequence of 26 kb 3' of the NEMO coding sequence is also present in the same position relative to the Delta NEMO locus, bringing the total length of the duplication to 35.5 kb. The LAGE2 gene is also located within this duplicated region, and a similar but unique LAGE1 gene is located just distal to the duplicated loci. Mapping and sequence information indicated that the duplicated regions are in opposite orientation. Analysis of the great apes suggested that the NEMO/LAGE2 duplication occurred after divergence of the lineage leading to present day humans, chimpanzees and gorillas, approximately 10-15 million years ago. Intriguingly, despite this substantial evolutionary history, only 22 single nucleotide differences exist between the two copies over the entire 35.5 kb, making the duplications >99% identical. This high sequence identity and the inverted orientations of the two copies, along with duplications of smaller internal sections within each copy, predispose this region to various genomic alterations. We detected four rearrangements that involved NEMO, Delta NEMO or LAGE1 and LAGE2. The high sequence similarity between the two NEMO/LAGE2 copies may be due to frequent gene conversion, as we have detected evidence of sequence transfer between them. Together, these data describe an unusual and complex genomic region that is susceptible to various types of pathogenic and polymorphic rearrangements, including the recurrent lethal deletion associated with IP.

Animals↗
PubMed1998

Structural analysis and evaluation of the aldosterone synthase gene in hypertension.

Anomalies in either of the tightly linked genes encoding the enzymes CYP11B1 (11beta-hydroxylase) or CYP11B2 (aldosterone synthase) can lead to important changes in arterial pressure and are responsible for several monogenically inherited forms of hypertension. Mutations in these genes or their regulatory regions could thus contribute to genetic variation in susceptibility to essential hypertension. To test this hypothesis, we performed 2 complementary studies of the CYP11B1/CYP11B2 locus in essential hypertension. After characterizing a DNA contig containing the CYP11B1 gene and mapping the gene in the Centre d'Etudes du Polymorphisme Humain reference panel of families, we performed a linkage study with 292 hypertensive sibling pairs and a highly informative microsatellite marker near CYP11B1. We also analyzed the association of 2 frequent biallelic polymorphisms of the CYP11B2 gene, 1 in the promoter at position -344 (-344C/T) and the other, a common gene conversion in intron 2, with hypertension in 380 hypertensive patients and 293 normotensive individuals. Statistical analyses did not show significant linkage of the CYP11B1 microsatellite marker to hypertension. No positive association with hypertension was found with the gene conversion in intron 2, but a positive association with hypertension was found with the -344T allele. The hypertensive and normotensive samples differed significantly in both genotype (P=0.023) and allele frequencies (P=0.010). Our data suggest a modest contribution of the CYP11B2 gene to essential hypertension.

Adult↗
PubMed1995

Detection and cloning of a common region of loss of heterozygosity at chromosome 1p in breast cancer.

The short arm of chromosome 1 is frequently affected by rearrangements in a variety of human malignancies. Genetic alterations, predominantly deletions, which are indicative of the presence of a putative tumor suppressor gene at chromosome 1p, are observed in breast cancer. In order to define the altered locus, eleven highly polymorphic microsatellite markers on chromosome 1p were used to detect loss of heterozygosity. We analyzed 52 cases of breast cancer and found 4 common deleted regions at chromosome 1p. Twenty-two of 52 (42%) informative patients showed at least 1 affected locus. The region most frequently exhibiting loss of heterozygosity was 1p31 (11/39; 28%); the other three common deleted regions were 1p36 (10/44; 23%), 1p35-36 (5/40; 13%), and 1p13 (8/39; 21%). These data suggest that one or more putative tumor suppressor genes may reside on chromosome 1p. We have cloned the entire region of interest at 1p31 in yeast artificial chromosomes. This yeast artificial chromosome contig can be used for fine mapping of the region and cloning of the candidate tumor suppressor gene.

Base Sequence↗
PubMed1996

Isolation of a cosmid clone corresponding to an inv(21) breakpoint of a patient with transient abnormal myelopoiesis.

Transient abnormal myelopoiesis (TAM) is a leukemoid reaction occurring occasionally on Down syndrome (DS) newborn infants. It has been hypothesized that "disomic homozygosity" in 21-trisomic cells plays an important role in the genesis of TAM, and the putative TAM gene was suggested to be mapped at a 21q11 region. We encountered a DS-associated TAM infant with a 47,XY,inv(21)(q11.1q22.13),+inv(21)(q11.1q22.13) karyotype. On the basis of another presumption that in this patient the putative TAM gene is disrupted by the break, we tried to isolate a breakpoint DNA. FISH analysis with cosmid clones corresponding to various sequence-tagged-site (STS) markers mapped at around 21q11.1-q11.2, we confirmed that the proximal breakpoint of the inv(21) was located between two STSs, G51E07 and D21S215, the latter locus being consistent with the previous tentative mapping. After construction of a cosmid contig encompassing between the two markers, we have isolated a cosmid clone corresponding to the proximal breakpoint of the inversion. This breakpoint was located near a previously identified duplicated region that is homologous to the sequence at 21q22.1. The isolated cosmid clone is useful for analysis of other TAM patients and for a search for a transcript at or flanking the breakpoint.

Adult↗
PubMed1997

A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes.

By searching the expressed sequence tag (EST) database, we identified partial cDNA sequences encoding a polypeptide with significant sequence identity to the human CC chemokine macrophage-inflammatory protein-1 alpha (MIP-1 alpha)/LD78 alpha. We determined the complete cDNA sequence that contained a reading frame of 89 amino acids with 61% identity to human MIP-1 alpha/LD78 alpha. The mRNA was expressed constitutively at high levels in human lung and at low levels in some lymphoid tissues. Furthermore, the mRNA was strongly induced in several human cell lines, including monocytic U937 cells, by PMA. From these results, we designated this novel CC chemokine as PARC from pulmonary and activation-regulated chemokine. In situ hybridization analyses showed that alveolar macrophages, follicular dendritic cells in the germinal centers of regional lymph nodes, and peripheral blood monocytes stimulated with LPS express PARC mRNA. Using the human CC chemokine yeast artificial chromosome contig that we constructed recently, we mapped the PARC gene (SCYA18) within one of the two subregions of the CC chemokine gene cluster at chromosome 17q11.2. To investigate its biologic activity, the PARC protein was expressed in insect cells. PARC was chemotactic for both activated (CD3+) T cells and nonactivated (CD14-) lymphocytes, but not for monocytes or granulocytes. Binding analysis using PARC fused with alkaline phosphatase-(His)6 showed the presence of a single class of receptors for PARC on lymphocytes with a Kd of 1.9 nM and 590 sites/cell. Thus, PARC is a novel CC chemokine with a close phylogenic relationship with MIP-1 alpha/LD78 alpha, but with a highly selective activity on lymphocytes.

Amino Acid Sequence↗
PubMed1996

The genes encoding the human CC-chemokine receptors CC-CKR1 to CC-CKR5 (CMKBR1-CMKBR5) are clustered in the p21.3-p24 region of chromosome 3.

The five human CC-chemokine receptors functionally characterized to date were mapped by using a radiation hybrid panel and YAC contigs. The genes encoding CC-CKR1, CC-CKR2, CC-CKR3, and CC-CKR5 (designated respectively CMKBR1, CMKBR2, CMKBR3, and CMKBR5 in the Genome Data Bank) were found to be clustered in the 3p21.3 region of chromosome 3, between the AFM362WB9 and the WI-6983 markers. The four genes fall within a total distance of about 350 kb. The fifth gene (CMKBR4, encoding the CC-CKR4 receptor) was located more distally (3p24) on the same chromosome, between the FB18G7 and the D3S1768 markers. These localizations were confirmed by mapping the genes into the YAC contigs covering these regions. The clustering of chemokine receptor genes suggests a relatively recent expansion of the gene family by gene duplication. Deletions and duplications of the 3p21 region have been described in neoplastic disorders of the hematopoietic lineage, suggesting a potential link with the CC-chemokine receptor gene family.

Chromosome Mapping↗
PubMed2000

The region on 9p associated with 46,XY sex reversal contains several transcripts expressed in the urogenital system and a novel doublesex-related domain.

Deletions of 9p have been associated with 46,XY gonadal dysgenesis, and the smallest region of overlap has been mapped to the tip of chromosome 9. Two candidate genes (DMRT1 and 2) have been found in the region. Despite intensive mutation searches, no mutations have been detected in these genes. To gain insights into the genomics of the region and to isolate other candidate genes for the phenotype, we have constructed a P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning over 500 kb and covering the consensus critical region. We have analyzed the expression pattern of several ESTs mapped or sublocalized within the framework of the contig. In addition, a sample shotgun sequencing of a PAC containing the mentioned DM genes led to the detection of novel transcripts displaying an expression pattern specific to testis and kidney, consistent with a role in the development of the urogenital system. One of them, expressed in adult testis and human embryos aged 4-5 weeks, encodes a potential polypeptide and is located immediately downstream of a sequence capable of encoding a novel DM domain. The region was partially screened for mutations in sex-reversed patients by Southern blot, sequencing, and FISH. No mutations were found. Our results suggest that the critical region on 9p involved in male-to-female sex reversal displays greater gene density and genomic complexity than previously anticipated. Future investigations will include functional and mutational studies of the novel transcripts mapped or sublocalized within the critical region by this study as well as cloning efforts to isolate additional candidate genes.

Adult↗
PubMed2002

[Molecular cloning of the human CX 58 gene].

OBJECTIVE: To clone a novel human connexin gene and find out the relationship between this gene and hereditary deafness. METHODS: Through the basic local alignment search tool (BLAST) analysis against the database of expressed sequence tags (dbEST) of National Center for Biotechnology Information (NCBI) using the coding sequence of mouse Cx 57 gene, 9 novel ESTs were obtained and a contig was assembled. Nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) were performed using primers designed on the contig. A novel gene was obtained and was mapped by homologous analysis against human genome sequence. Mutation analysis was performed in 12 autosomal dominant hereditary deafness families. RESULTS: Nine ESTs were obtained by homologous analysis and a contig was assembled. Through nested PCR and RACE, a full length of cDNA was obtained from human liver, kidney Ready cDNA and placenta cDNA library, and was named CX 58. By comparison with human genome sequence, CX 58 was mapped at 1p32.3-p34.1. Mutation analysis of CX 58 was performed in 12 autosomal dominant hereditary deafness families, but no mutation was detected. CONCLUSION: A novel human connexin gene named CX 58 was cloned and mapped to 1p32.3-p34.1. The mutation of CX 58 may not result in autosomal dominant hereditary deafness.

Animals↗
PubMed2001

A BAC-based STS-content map spanning a 35-Mb region of human chromosome 1p35-p36.

We have devised a mapping method for rapid assembly and ordering of bacterial artificial chromosome (BAC) clones on a radiation hybrid (RH) panel, using sequence-tagged sites (STSs) and PCR. The protocol consists of two rounds of two-dimensional screening from a limited number of BACs to correspond each to an STS. In the first round, STSs are assembled in the RH bins and ordered according to PCR signals derived from 384-well microtiter plates (MTPs) in which BAC clones have been arrayed. In the second round, individual BAC clones are isolated from the MTPs to build a contig. We applied this method to a 35-Mb region spanning human chromosome 1p35-p36 and assembled 1366 BACs in 11 contigs, the longest being about 20 Mb. The working draft sequences of the human genome have been integrated into the contigs to validate the accuracy.

Chromosome Mapping↗
PubMed1997

Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome.

The positioning of Expressed Sequence Tags (ESTs) constitutes an important step towards a functional map of the human genome, including candidate genes for human genetic disorders that have been localized by linkage analysis. We localized 59 ESTs on the human X chromosome, including 44 derived from infant brain and 15 from adult muscle cDNA libraries. Localizations by a somatic cell hybrid panel were refined for five cDNAs by mapping them in yeast artificial chromosome (YAC) contigs.

Animals↗
PubMed2004

Chromosome walking in the Petunia inflata self-incompatibility (S-) locus and gene identification in an 881-kb contig containing S2-RNase.

Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is controlled by the polymorphic S locus, which contains two separate genes encoding pollen and pistil determinants in SI interactions. The S-RNase gene encodes the pistil determinant, whereas the pollen determinant gene, named the pollen S gene, has not yet been identified. Here, we set out to construct an integrated genetic and physical map of the S locus of Petunia inflata and identify any additional genes located at this locus. We first conducted chromosome walking at the S2 locus using BAC clones that contained either S2-RNase or one of the nine markers tightly linked to the S locus. Ten separate contigs were constructed, which collectively spanned 4.4 Mb. To identify additional genes located at the S2 locus, a 328-kb region (part of an 881-kb BAC contig) containing S2-RNase was completely sequenced. Approximately 76% of the region contained repetitive sequences, including transposon-like sequences. Other than S2-RNase, an F-box gene, named PiSLF2 (S2-allele of P. inflata S-locus F-box gene), was the only predicted gene whose deduced amino acid sequence was similar to the sequences of known proteins in the database. Two different cDNA selection methods were used to identify additional genes in the 881-kb contig; 11 groups of cDNA clones were identified in addition to those for S2-RNase and PiSLF2. RT-PCR analysis of expression profiles and PCR analysis of BAC clones and genomic DNA confirmed that seven of these 11 newly identified genes were located in the 881-kb contig.

Chromosome Walking↗
PubMed1992

Integration of gene maps: chromosome 1.

A composite map of 177 loci has been constructed in two steps. The first combined pairwise logarithm-of-odds scores on 127 loci into a comprehensive genetic map. Then this map was projected onto the physical map through cytogenetic assignments, and the small amount of physical data was interpolated for an additional 50 loci each of which had been assigned to an interval of less than 10 megabases. The resulting composite map is on the physical scale with a resolution of 1.5 megabases. In the future these methods may be used to incorporate locations from linkage, contigs, radiation hybrids, restriction fragments, and somatic cell maps. Dense, reliable, and well-documented maps are essential for long-range sequencing and to localize and clone disease genes.

Animals↗