An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: Isolation of SfiI linking clones from a chromosome 21-specific library

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking gragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.     

A novel procedure for selective cloning of NotI linking fragments from mammalian genomes.

A novel procedure has been developed for selective cloning of NotI linking fragments from mammalian genomes. Since the majority of the NotI sites in mammalian genomes are considered to be localized in so-called HTF (HpaII tiny fragment) islands, an HTF library was constructed as an initial step to enrich the NotI sites. The plasmid DNAs were isolated en masse from the HTF library and digested with NotI. Linearized plasmid DNAs derived from NotI linking clones were efficiently separated from undigested circular DNAs by an unique pulsed field polyacrylamide gel electrophoresis (PF-PAGE). The linear DNAs were eluted from the gel, recircularized with T4 DNA ligase and introduced into E. coli cells. About 95% of the transformants were found to contain NotI linking fragments. The procedure will thus provide a simple and useful way of collecting NotI linking fragments for long range physical mapping of mammalian genomes.     

Agarose entrapment method for the production of SfiI linking library for Theileria parva

We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhanging ends that preclude the direct ligation into vector sites required by most alternative strategies, but that favor the recircularization reactions used here.     

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

Identification of a 220-kb insertion into the Duchenne gene in a family with an atypical course of muscular dystrophy

Most known mutations in the gene region responsible for Duchenne or Becker muscular dystrophy are deletions of varying extent. Here we describe a 220-kb insertion within the DMD/BMD gene that cosegregates with a somewhat atypical course of muscular dystrophy in a pedigree. The insertion is demonstrated by field-inversion gel electrophoresis as an enlarged SfiI fragment hybridizing to probe J-Bir, while neighboring SfiI fragments (detected by probes PERT 87 and J-66) are unchanged. Hybridization with DMD c-DNA probes did not reveal alterations in coding sequences. In this pedigree, the altered SfiI fragments provide convenient markers for carrier identification.     

Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells

A method was developed for amplification and expression of foreign genes in mammalian cells. This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent. Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos. The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells. Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites. A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively. The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1. A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli. Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids. More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and characterization of a NotI linking library of human chromosome 21

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.     

Construction of a chromosome 15-specific linking library and identification of potential gene sequences

We have tested a strategy for the construction of a total restriction fragment map of a human chromosome and the rapid isolation of a great number of genes from a specific chromosome. The strategy is based on the cloning of chromosome-specific CpG-rich DNA sequences which are present at the 5' end of many genes. This approach has two important implications: (i) the clones can be used to probe pulsed-field gradient gel blots and link restriction fragments generated by CpG restriction endonucleases, and (ii) the finding of tight genetic or physical linkage between one of these gene probes and a given hereditary disease would make the marker a good candidate gene for this disease. We have constructed a chromosome 15-specific linking library and identified potential gene sequences.     

Physical analysis and mapping of the Mycoplasma pneumoniae chromosome.

Field inversion gel electrophoresis was used for analysis of the chromosome of Mycoplasma pneumoniae. The restriction endonuclease SfiI (5'-GGCCNNNNNGGCC-3') generated 2 M. pneumoniae DNA fragments of approximately 437 and 357.5 kilobase pairs (kbp), whereas 13 restriction fragments ranging in size from 2.4 to 252.0 kbp resulted from digestion with ApaI (5'-GGGCCC-3'). Totaling the sizes of the individual restriction fragments from digestion with SfiI or ApaI yielded a genome size of 794.5 or 775.4 kbp, respectively. A physical map of the M. pneumoniae chromosome was constructed by using a combination of techniques that included analysis by sequential or partial restriction endonuclease digestions and use as hybridization probes of cloned M. pneumoniae DNA containing ApaI sites and hence overlapping adjacent ApaI fragments. Genetic loci for deoC, rrn, hmw3, and the P1 gene were identified by using cloned DNA to probe ApaI restriction fragment profiles.     

Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis.

Of 16 restriction endonucleases known to hydrolyze rare 6- or 8-base recognition sequences that were tested, only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Yersinia pestis, the causative agent of bubonic plague, of sufficient length to permit physical analysis by pulsed-field gel electrophoresis (PFGE). Of the individual bands detected after single-dimensional PFGE of these digests, the largest sum was obtained with SpeI (3,575.6 +/- 114.6 kb). Of these 41 bands, 3 were found to contain comigrating fragments, as judged by the results of two-dimensional SpeI-ApaI PFGE; addition of these fragments and the three plasmids of the species yielded a refined estimate of 4,397.9 +/- 134.6 kb for the genome. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. The high-frequency chromosomal deletion known to exist in nonpigmented mutants (unable to assimilate Fe3+ at 37 degrees C or store hemin at 26 degrees C) was shown by two-dimensional PFGE analysis with SpeI and ApaI or with SfiI and SpeI to be 92.5 and 106 kb in size, respectively. The endpoints of this deletion were precise, and its size was more than sufficient to encode the eight known peptides reported to be absent in nonpigmented mutants. This deletion had not occurred (but was able to do so) in a rare mutant capable of hemin storage but not iron transport.     

Physical and genetic chromosomal map of an M type 1 strain of Streptococcus pyogenes.

A physical map of the chromosome of an M type 1 strain of Streptococcus pyogenes was constructed following digestion with three different restriction enzymes, SmaI, SfiI, and SgrAI, and separation and analysis of fragments by pulsed-field gel electrophoresis. The genome size of this strain was estimated to be 1,920 kb. By employing Southern hybridization and PCR analysis, 36 genes were located on the map.     

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.     

A method for two-dimensional DNA electrophoresis (2D-DE): application to the immunoglobulin heavy chain variable region

The scarcity of single-copy probes creates difficulty in the generation of large-scale physical maps of mammalian gene families. A simple method of two-dimensional DNA electrophoresis (2D-DE) has been developed to overcome this problem. DNA (2 micrograms) is digested with a rare-cutting restriction endonuclease and size separated by pulsed-field gel electrophoresis (PFGE). The DNA, still contained within the lane of the PFGE gel, is digested with a second frequent-cutting restriction enzyme and is subjected to an electrical field perpendicular to that of the PFGE. 2D-DE allows the simultaneous mapping, to large restriction fragments, of all the genes detected by a particular probe. The human immunoglobulin variable region was used as an example for this procedure. Two VH5 genes, on 8- and 9-kb EcoRI fragments, were mapped to 200- and 65-kb SfiI fragments, respectively, by 2D-DE. This technique will be particularly useful in the generation of physical maps of complex human gene families and of repeat families.     

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

Molecular analysis of the human MHC class I region using yeast artificial chromosome clones

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.     

Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes.

A linking library consists of genomic DNA fragments which contain a specific rare restriction enzyme site; such clones are very useful as probes in pulsed field gel electrophoresis and in mapping and cloning large regions of DNA. However, identifying those linking clones which map to a certain chromosomal region can be laborious. Therefore, we have developed a straightforward procedure for constructing a linking library directly from flow-sorted chromosomes. As a test of the approach, a NotI linking library was constructed from the chromosome 17 fraction of a flow-sort of human chromosomes, using only 70 ng of DNA. Thirteen of sixteen linking clones were mapped to chromosome 17, suggesting that the library is highly enriched for this chromosome. This method should be generally applicable to other chromosomes and enzymes as well.     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.