A cosmid and yeast artificial chromosome contig containing the complete ryanodine receptor (RYR1) gene

The ryanodine receptor (RYR1) gene is responsible for some forms of malignant hyperthermia and has been localized to 19q13.1. Central core disease is a genetic myopathy that is genetically linked to RYR1. We have identified an overlapping set of cosmid and YAC clones that spans more than 800 kb and includes the RYR1 gene (approximately 205 kb). Cosmids from this region were identified by screening three chromosome 19 cosmid libraries (11-fold coverage) with six subclones representing the entire RYR1 cDNA. Genomic sequences from positive cosmids were then used as probes to identify additional cosmids. A minimally overlapping set of 23 cosmids was assembled into two contigs on the basis of restriction fragment analysis and hybridization data. Three YAC clones were isolated by screening a human YAC library with selected cosmid inserts. Overlaps among these YACs and the cosmid contigs were determined by hybridizing YAC Alu-PCR products to cosmid DNAs. The YACs bridged the gap between the cosmid contigs and extended the contig on both sides. Fluorescence in situ hybridization experiments positioned the RYR1 contig between GPI, MAG, and D19S191 on the proximal side and D19S190, CYP2A, CYP2F, SNRPA, BCKDHA, and other markers on the distal side. The 800-kb contig of cloned reagents will facilitate the detailed characterization of the RYR1 gene and other loci that may be closely related to central core disease.     

Human immunodeficiency virus type 1 may preferentially integrate into chromatin occupied by L1Hs repetitive elements

Human DNA flanking sites of eight human immunodeficiency virus type 1 (HIV-1) proviral integrations have been analyzed in isolates derived both from integrations in an infected individual and from tissue culture. Sequence analysis encompassing 80-3000 bp of human DNA on one or both sides of the site of integration revealed that seven of the eight HIV-1 proviruses had integrated directly into or within one nucleosome's distance from an L1Hs or Alu repetitive element. To compare this with the frequency at which human L1 or Alu elements sharing > or = 70% identity with L1Hs and Alu consensus sequences would be encountered at random, > 200 bp from each of 82 individual anonymously cloned segments of human DNA were sequenced: L1Hs elements were encountered in 8.5% of the 82 clones and Alu elements were encountered in 13.4+ by using these homology windows. From these data it appears that HIV-1 integrates into or near L1Hs elements with an approximately 6-fold higher frequency than would be expected if HIV-1 integration events were distributed uniformly throughout the genome. A cumulative binomial probability test shows that there is a 0.26% chance that one would arrive at these figures by chance and puts the data well within a 99% confidence interval. We propose that sites of L1Hs and Alu insertions originally occurred in regions of chromatin that were more easily accessible to the retroposon machinery and that these regions are now acting as preferred integration sites for HIV-1.     

The evolution of MHC diversity by segmental duplication and transposition of retroelements

Sequence analysis of a 237 kb genomic fragment from the central region of the MHC has revealed that the HLA-B and HLA-C genes are contained within duplicated segments peri-B (53 kb) and peri-C (48 kb), respectively, and separated by an intervening sequence (IF) of 30 kb. The peri-B and peri-C segments share at least 90% sequence homology except when interrupted by insertions/deletions including Alu, L1, an endogenous retrovirus, and pseudogenes. The sequences of peri-B, IF, and peri-C were searched for the presence of Alu elements to use as markers of evolution, chromosomal rearrangements, and polymorphism. Of 29 Alu elements, 14 were identified in peri-B, 11 in peri-C, and 4 in IF. The Alu elements in peri-B and peri-C clustered phylogenetically into two clades which were classified as "preduplication" and "postduplication" clades. Four Alu J elements that are shared by peri-B and peri-C and are flanked by homologous sequences in their paralogous locations, respectively, clustered into a "preduplication" clade. By contrast, the majority of Alu elements, which are unique to either peri-B or peri-C, clustered into a postduplication clade together with the Alu consensus subfamily members ranging from platyrrhine-specific (Spqxcg) to catarrhine-specific Alu sequences (Y). The insertion of platyrrhine-specific Alu elements in postduplication locations of peri-B and peri-C implies that these two segments are the products of a duplication which occurred in primates prior to the divergence of the New World primate from the human lineage (35-44 mya). Examination of the paralogous Alu integration sites revealed that 9 of 14 postduplication Alu sequences have produced microsatellites of different length and sequence within the Alu 3'-poly A tail. The present analysis supports the hypothesis that HLA-B and HLA-C genes are products of an extended segmental duplication between 44 and 81 million years ago (mya), and that subsequent diversification of both genomic segments occurred because of the mobility and mutation of retroelements such as Alu repeats.     

Creation of a deletion series of mouse YACs covering a 500 kb region around Xist

Two mouse YACs, PA-2 and PA-3, contain the Xist gene and are 460 kb and 3.3 Mb long respectively. While PA-2 is non-chimeric, PA-3 contains a substantial proportion of non-contiguous DNA. As a prerequisite to functional studies of the role of this region in X inactivation, we have created a deletion series of YACs that are spaced at approximately 50 kb intervals and were able to eliminate the unwanted chimeric sequences in YAC PA-3. For this purpose, we have constructed mouse B1 fragmentation vectors based on those described for human Alu fragmentation. Having created this series of YAC deletion derivatives, we were able to eliminate efficiently the 10-15% aberrant YACs that arise during the course of a fragmentation experiment by assessing their marker content. The overlap and the opposite orientation of the two YAC inserts permitted the creation of deletions on both sides of the 500 kb region around Xist. The use of this series of YACs in a biological assay will help us define the extent of the sequences necessary to bring about X chromosome inactivation.     

Alu elements of the primate major histocompatibility complex

The chromosomal region constituting the major histocompatibility complex (MHC) has undergone complex evolution that is often difficult to decipher. An important aid in the elucidation of the MHC evolution is the presence of Alu elements (repeats) which serve as markers for tracing chromosomal rearrangements. As the first step toward the establishment of sets of evolutionary markers for the MHC, Alu elements present in selected MHC haplotypes of the human species, the gorilla, and the chimpanzee were identified. Restriction fragments of cosmid clones from the libraries of the three species were hybridized with Alu-specific probes, Alu elements were amplified by the polymerase chain reaction, and the amplification products were sequenced. In some cases, sequences of the regions flanking the Alu elements were also obtained. Altogether, 31 new Alu elements were identified, representing six Alu subfamilies. The average density of Alu elements in the MHC is one element per four kilobases (kb) of sequence. Alu elements have apparently been inserted steadily into the MHC over the last 65 million years (my). On average, one Alu element is inserted into the primate MHC every 4 my. Analysis of the human DR3 haplotype supports its origin by duplication from an ancestral haplotype consisting of DRB1 and DRB2 genes. The sharing of an old Alu element by the DRB1 and DRB2 genes, in turn, supports their divergence from a common ancestor more than 55 my ago.     

Sequence and evolutionary history of the length polymorphism in intron 1 of the human red photopigment gene

The length of intron 1 of the red pigment gene is dimorphic among Africans but not among Caucasians or Asians. This dimorphism was found to result from the presence or absence of a block of 1,284 bp comprised of three Alu elements and 328 bp of intervening unique-sequence DNA. This additional sequence in the "long" intron 1 of the red pigment gene was shown to have inserted into a fourth and older Alu element present in the "short" from of intron 1. Furthermore, the size and sequence of the "short" intron 1 of the red pigment gene is equivalent to that of the adjacent green pigment gene. The block containing the three Alu elements was not found in intron 1 of the red or green pigment genes of Old World monkeys and orangutans but was present in intron 1 of both the green and red pigment genes of gorillas and chimpanzees. The nucleotide sequence of this block in Old World primates and the estimated ages of the three elements suggest that their insertion occurred sequentially in the Old World monkey lineage prior to duplication of the ancestral X-chromosome-linked pigment gene. After gene duplication, deletion of the entire block containing the three Alu elements from one of the genes created the "short" intron variant. Unequal recombination between the adjacent and highly homologous red and green pigment genes may have resulted in the formation of the "short" intron variant of the other gene.     

Hemodynamic and cerebral blood flow effects of cocaine, cocaethylene and benzoylecgonine in conscious and anesthetized fetal lambs

Yeast artificial chromosome (YAC) clones propagate large segments of exogenous DNA in a host organism with well-developed classical and molecular genetics. Most extant YAC clones are from libraries created in a single yeast host (AB1380). The application of techniques allowing the manipulation and/or restructuring of these cloned DNA segments often requires a change in the yeast genetic background to introduce desirable genetic markers. Transfer methods in current use require extremely high yeast transformation efficiencies or require access to equipment for yeast tetrad analysis. We have developed an alternative method for moving YAC clones from one yeast strain to another, taking advantage of the properties of kar1 mutants altered in a gene required for normal karyogamy (nuclear fusion) during mating. Transfer by this method requires generally accessible methods, including yeast cell culture, replica plating, and pulsed-field gel electrophoresis. We present data demonstrating efficient transfer of nine different YACs from their original host (AB1380) to a kar1 recipient strain (YPH925) with genetic markers that facilitate the use of existing homologous recombination-based modification methods. The enhanced ability to transfer clones to this new host will accelerate the pace of refinement and fine-structure mapping of the YAC contigs currently under construction and facilitate gene manipulation on YACs for subsequent functional analysis.     

Human chromosome 5 sequence primer amplifies Alu polymorphisms on chromosomes 2 and 17

Members of the Alu family of repetitive elements occur frequently in the human genome and are often polymorphic. Techniques involving Alu element mediated polymerase chain reactions (Alu PCR) allow the isolation of region-specific human DNA fragments from mixed DNA sources. Such fragments are a source of region-specific Alu elements useful for the detection of Alu-related polymorphisms. A clone from human chromosome 5, corresponding to locus D5F40S1, was isolated using Alu PCR differential hybridization. Alu elements within this clone were investigated for the presence of potentially polymorphic 3' polyA tails. Primers were devised to amplify the 3' polyA tail of an Alu element present within the clone. One primer, D5F40S1-T, was specific to the DNA flanking the 3' end of the Alu element, and the other primer was homologous to sequences within the element. When these primers were used in PCR reactions, products from chromosomes 2 and 17 (loci D2F40S2 and D17F40S3) were amplified in addition to the expected product from chromosome 5. The most likely explanation for this nonspecific amplification is that the D5F40S1-T primer is located within a low-copy repetitive element that is 3' of the Alu element. This phenomenon presents a potential problem for the identification of region-specific Alu polymorphisms.     

Autoradiographic and SPECT imaging of cerebral opioid receptors with an iodine-123 labeled analogue of diprenorphine

To get insight into the early evolution of the primate Alu elements, we characterized sequences of these repeats from the Malagasy prosimians, lemurs (Lemuridae) and sifakas (Indriidae), as well as from galagos (Lorisidae). These sequences were compared with the oldest Alu species known from the human genome: dimeric Alu J and S and free Alu monomers. Our analysis indicates that about 60 Myr ago, before the prosimian divergence, free left and right monomers formed an Alu heterodimer connected by a 19-nucleotide-long A-rich linker. The resulting elements successfully propagated in diverging primate lineages until about approximately 20 Myr ago, conserving similar sequence features and essentially the same Alu RNA secondary structure. We suggest that until that time the same "retropositional niche", molecular machinery making possible the proliferation by retroposition, constrained the evolution of Alu elements in extant primate species. These constraints became subsequently relaxed. In the Malagasy prosimians the dimeric Alu continued to amplify after acquiring a 34- to 36-nucleotide extension of their linker segment, whereas in the galago genome the "retropositional niche" was occupied by novel short elements.     

Construction of YAC contigs on rice chromosome 5

A physical map of rice chromosome 5 was constructed with yeast artificial chromosome (YAC) clones along a high-resolution molecular linkage map carrying 118 DNA markers distributed over 123.7 cM of genomic DNA. YAC clones have been identified by colony and Southern hybridization for 105 restriction fragment length polymorphism (RFLP) markers and by polymerase chain reaction (PCR) screening for 8 sequence-tagged site (STS) markers and 5 randomly amplified polymorphic DNA (RAPD) markers. Of 458 YACs, 235 individual YACs with an average insert length of 350 kb were selected and ordered on chromosome 5 from the YAC library. Forty-eight contigs covering nearly 21 Mb were formed on the chromosome 5; the longest one was 6 cM and covered 1.5 Mb. The length covered with YAC clones corresponded to 62% of the total length, of chromosome 5. There were many multicopy sequences of expressed genes on chromosome 5. The distribution of many copies of these expressed gene sequences was determined by YAC Southern hybridization and is discussed. A physical map with these characteristics provides a powerful tool for elucidation of genome structure and extraction of useful genetic information in rice.     

Inactivation of the p53 tumor suppressor gene via a novel Alu rearrangement

Inactivation of the p53 tumor suppressor gene is a common finding in human cancer. In most cases, inactivation is due to a point mutation in the gene, but rearrangement of the p53 gene is sometimes observed. We analyzed the inactivation of p53 in the human pancreas cancer cell line Hs766T, which harbors a structural alteration in the p53 gene. This inactivation was found to be the result of a complex deletion/insertion event involving at least two different Alu elements. The rearrangement eliminated exons 2-4 from the p53 gene, whereas a 175-bp Alu fragment was inserted between the breakpoints of the deletion. DNA sequence analysis of this Alu fragment revealed that it is identical to an Alu element in intron 1 of the p53 gene. This is the first report of p53 inactivation due to a rearrangement involving Alu elements. This type of inactivation may go unnoticed when only traditional methods to detect p53 alterations are used.     

Subtracted, unique-sequence, in situ hybridization: experimental and diagnostic applications

Nonrandom chromosomal aberrations, particularly in cancer, identify pathogenic biological pathways and, in some cases, have clinical relevance as diagnostic or prognostic markers. Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical and structural chromosome abnormalities. We report the development of robust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of adapter-ligated, short-fragment, DNA libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybridization. These subtracted probes are labeled conveniently, and the fluorescence or colorimetric detection signals are extremely bright. Moreover, they constitute a stable resource that may be amplified through at least four rounds of polymerase chain reaction without diminishing signal intensity. We demonstrate applications of subtracted probes for the MYC and EWS oncogene regions, including 1) characterization of a novel EWS-region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identification of chromosomal translocations by conventional bright-field microscopy.     

Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.     

Balloon aortic valvuloplasty

Sequence-tagged site (STS) content mapping in yeast artificial chromosomes (YACs) was used to cover the region deleted in two patients affected with X-linked lymphoproliferative disorder. The order of markers includes, centromere to telomere, DXS8009-DXS1206-DXS8078-DXS8044-DXS982- DXS6811-DXS8093-AFM240xblO- DXS75-DXS737-DXS100-DXS6-DXS1046-DXS803 8. The order of six major markers is confirmed by fluorescent in situ hybridization, and all the markers assigned by linkage mapping fall within a 1.6-cM interval. The contig comprises 90 clones containing 89 STSs, yielding a resolution of 50 kb; DNA in a gap just telomeric to DXS8044 has not been found in > 20 equivalents of YACs or bacterial clones. The two deletions were found to have centromeric breakpoints that lie close to DXS1206 and may be identical; the telomeric breakpoints are -150 kb apart, one falling between DXS737 and DXS100, the other between DXS100 and DXS1046. Several STSs near the breakpoints show weak amplification from more than one site; one gives products from three groups of YACs, and lie, respectively, within 50 kb of the centromeric and the two telomeric deletion borders. Such partially duplicated segments of DNA are candidates for involvement in the formation of the deletions.     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.