Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.     

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.     

Construction and analysis of linking libraries from the mouse X chromosome

A hybrid cell line containing the mouse X chromosome on a human background has been used to construct linking libraries from the mouse X chromosome, and approximately 250 unique EagI and NotI clones have been identified. Seventy-three clones have been sublocalized onto the X chromosome using interspecific Mus spretus/Mus domesticus crosses and a panel of somatic cell hybrids carrying one-half of reciprocal X-autosome translocations. The average spacing of the linking clones mapped to date is about one every 2 Mb of DNA. Two clones from the central region of the chromosome have been physically linked by pulsed-field gel electrophoresis. A large number of clones contain conserved sequences, indicating the presence of CpG-rich island-associated genes. The clones isolated from these libraries provide a valuable resource for comparative mapping between man and mouse X chromosomes, isolation of X-linked disease loci of interest by reverse genetics, and analysis of the long-range structure and organization of the chromosome.     

Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.     

Isolation of repetitive DNA sequences from human chromosome 21.

We have developed a method for the isolation of phage from the human genomic library that carry repetitive DNA sequences highly represented on specific human chromosomes. We have used this technique to select recombinants carrying inserts concentrated on chromosome 21. Five clones, representing two families of sequences, have been characterized. Members of each family show cross-homology, but the two families show no homology with each other. In all but one case, the clones do not contain members of the human Alu repeat family. Single chromosome-concentrated repetitive sequences should prove to be useful in studies of the origin, evolution, and function of repetitive DNA and in regional chromosome mapping.     

The FSHD-Associated Repeat, D4Z4, Is a Member of a Dispersed Family of Homeobox-Containing Repeats, Subsets of Which Are Clustered on the Short Arms of the Acrocentric Chromosomes

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35. FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4. D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs. In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease. However, no expressed sequences from D4Z4 have been identified. We have previously shown that there are other loci similar to D4Z4 within the genome. In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat. Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes. D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies. The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed. We propose the name 3.3-kb repeat for this family of repetitive sequence elements.     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

An unusual Alu repeat sequence within the CAD gene

Summary There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangments may be a factor in generating the diversity of Alu repeats found in primate genomes.     

Evolution of alu family repeats since the divergence of human and chimpanzee

Summary The DNA sequences of three members of the Alu family of repeated sequences located 5 to the chimpanzee 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.     

Human repeat element-mediated PCR: cloning and mapping of chromosome 10 DNA markers.

Repeat element-mediated PCR can facilitate rapid cloning and mapping of human chromosomal region-specific DNA markers from somatic cell hybrid DNA. PCR primers directed to human repeat elements result in human-specific DNA synthesis; template DNA derived from a somatic cell hybrid containing the human chromosomal region of interest provides region specificity. We have generated a series of repeat element-mediated PCR clones from a reduced complexity somatic cell hybrid containing a portion of human chromosome 10. The cloning source retains the centromere and tightly linked flanking markers, plus additional chromosome 10 sequences. Twelve new inter-Alu, two inter-L1, and four inter-Alu/L1 repeat element-mediated PCR clones were mapped by hybridization to Southern blots of repeat element-mediated PCR products amplified from somatic cell hybrid DNA templates. Two inter-Alu clones mapped to the pericentromeric region. We propose that a scarcity of Alu elements in the pericentromeric region of chromosome 10 contributed to the low number of clones obtained from this region. One inter-Alu clone, pC11/A1S-6-c23, defines the D10S94 locus, which is tightly linked to MEN2A and D10Z1.     

Chromosome-specific subfamilies within human alphoid repetitive DNA

Nucleotide sequence data of about 20 X 10(3) base-pairs of the human tandemly repeated alphoid DNA are presented. The DNA sequences were determined from 45 clones containing EcoRI fragments of alphoid DNA isolated from total genomic DNA. Thirty of the clones contained a complete 340 base-pair dimer unit of the repeat. The remaining clones contained alphoid DNA with fragment lengths of 311, 296, 232, 170 and 108 base-pairs. The sequences obtained were compared with an average alphoid DNA sequence determined by Wu & Manuelidis (1980). The divergences ranged from 0.6 to 24.6% nucleotide changes for the first monomer and from 0 to 17.8% for the second monomer of the repeat. On the basis of identical nucleotide changes at corresponding positions, the individual repeat units could be shown to belong to one of several distinct subfamilies. The number of nucleotide changes defining a subfamily generally constitutes the majority of nucleotide changes found in a member of that subfamily. From an evaluation of the proportion of the total amount of alphoid DNA, which is represented by the clones studied, it is estimated that the number of subfamilies of this repeat may be equal to or exceed the number of chromosomes. The expected presence of only one or a few distinct subfamilies on individual chromosomes is supported by the study, also presented, of the nucleotide sequence of 17 cloned fragments of alphoid repetitive DNA from chromosome 7. These chromosome-specific repeats all contain the characteristic pattern of 36 common nucleotide changes that defines one of the subfamilies described. A unique restriction endonuclease (NlaIII) cleavage site present in this subfamily may be useful as a genetic marker of this chromosome. A family member of the interspersed Alu repetitive DNA was also isolated and sequenced. This Alu repeat has been inserted into the human alphoid repetitive DNA, in the same way as the insertion of an Alu repeat into the African green monkey alphoid DNA.     

Interspersed repeated sequences in the African green monkey genome that are homologous to the human Alu family.

The dominant family of interspersed repetitive DNA sequences in the human genome has been termed the Alu family. We have found that more than 75% of the lambda phage in a recombinant library representing an African green monkey genome hybridize with a human Alu sequence under stringent conditions. A group of clones selected from the monkey library with probes other than the Alu sequence were analyzed for the presence and distribution of Alu family sequences. The analyses confirm the abundance of Alu sequences and demonstrate that more than one repeat unit is present in some phages. In the clones studied, the Alu units are separated by an average of 8 kilobase pairs of unrelated sequences. The nucleotide sequence of one monkey Alu sequence is reported and shown to resemble the human Alu sequences closely. Hence, the sequence, dispersion pattern, and copy number of the Alu family members are very similar in the African green monkey and human genomes. Among the clones investigated were two that contain segments of the satellite DNA term alpha-component joined to non alpha-component DNA. The experiments indicate that in the monkey genome Alu sequences can occur close to regions of alpha-component DNA.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background

A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.     

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 bromide-stained 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.     

The adult α-globin locus of old world monkeys: An abrupt breakdown of sequence similarity to human is defined by an Alu family repeat insertion site

Summary The haploid genomes of all known primates have two or more adult -globin genes contained within tandemly arranged duplication units. Although the tandem duplication event generating these -globin loci is believed to occur prior to the divergence of primates, a number of length polymorphisms exist within the loci among different primate species. In order to understand the molecular basis of these length polymorphisms, we have cloned and determined the nucleotide sequence of a major portion of the rhesus monkey adult -globin locus. Sequence comparison to human suggests that the length difference between the adult -globin loci of human and Old World monkey is the result of one or more DNA recombination processes, all of which appeared to be related to the transposition of Alu family repeats. First, the finding of a monomeric Alu family repeat at the junction between nonhomology block I and homology block Y of the 2 genecontaining unit in rhesus macaque suggests that the dimeric Alu family repeat, Alu 3, at the orthologous position in human was generated by insertion of a monomeric Alu family repeat into the 3 end of another preexisting Alu family repeat. Second, two Alu family repeats, Alu 1 and Alu 2, exist in human at the 3 end of each of the two X homology blocks, respectively. However, this pair of paralogous Alu family repeats is absent at the corresponding positions in rhesus macaques. This raises interesting questions regarding the evolutionary origin of Alu 1 and Alu 2. Finally, DNA sequences immediately downstream from the insertion site of Alu 2 are completely different between human and rhesus macaque. This last event is similar to DNA rearrangements occurring nearby transposable element(s) in the chromosomes of bacteria, yeast, and plant cells. Its possible role in accelerating the genomic evolution of noncoding or spacer DNA is discussed.     

Presence and phylogenetic analysis of HERV-K LTR on human X and Y chromosomes: evidence for recent proliferation

The K group of human endogenous retroviruses (HERV-K) has been suggested to have a role in disease and has recently been shown to include long terminal repeat (LTR) elements that are human specific. Here we investigated the presence of HERV-K LTRs on the human X and Y chromosomes with the use of PCR on a monochromosomal somatic cell hybrid DNA panel. We report twelve such sequences on the X chromosome and ten sequences on the Y chromosome. Phylogenetic analysis reveals that clones X2, 4, 5, 6, 7, 11, 15 from the X chromosome and clones Y4, 5, 7, 10 from the Y chromosome are closely related to the human-specific members of Medstrand and Mager's cluster 9. The sequence of clone Y7 from the Y chromosome is identical with human-specific HERV-K LTR element (AC002350) from chromosome 12q24. The findings suggest recent proliferation and transposition of HERV-K LTR elements on these chromosomes. Such events may have contributed to structural change and genetic variation in the human genome. We draw attention to evolutionarily recent changes in homologies between X and Y chromosomes as a method of further investigating such transpositions.     

Variation in genomic alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human:rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse:human hybrid cells that span the entire long arm of human chromosome 17.by L. Manuelidis     

"PCR-karyotype" of human chromosomes in somatic cell hybrids

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes were performed by the polymerase chain reaction (PCR) using primer directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a "PCR-karyotype." This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosome produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.