Construction of Highly Extensive Polymorphic DNA Libraries by in-Gel Competitive Reassociation Procedure

Differential genomic DNA libraries between two mouse strains and from two human individuals were constructed by means of the in-gel competitive reassociation (IGCR) procedure, a procedure developed for cloning altered anonymous restriction fragments. The libraries were highly enriched in RFLP fragments, approximately 60 and 40% for the mouse and human libraries, respectively, and, more importantly, maintained most of the original complexities of the RFLP fragments. Therefore, differential genomic DNA libraries constructed by the IGCR procedure, particularly for human genomic DNA, should offer highly extensive sources for polymorphic DNA sequences necessary for a variety of genome analyses, including studies on the origin and mechanism of biological diversity among the same species.     

Intra-strain polymorphisms are detected but no genomic alteration is found in cloned mice

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Here, we applied the IGCR procedure to two cloned mice derived from an F1 hybrid of the C57BL/6Cr and DBA/2 strains, in order to investigate the possibility of genomic alteration in the cloned mouse genomes. Each of the five of the genomic alterations we detected between the two cloned mice corresponded to the "intra-strain" polymorphisms in the C57BL/6Cr and DBA/2 mouse strains. Our result suggests that no severe aberration of genome sequences occurs due to somatic cell nuclear transfer.     

Construction of Libraries for Methylation Sites by In-gel Competitive Reassociation (IGCR)

The in-gel competitive reassociation (IGCR) procedure was successfullyapplied to construct a comprehensive library enriched in DNAfragments containing C^5mCGG sequences from mouse liver and braingenomic DNA. For IGCR, methylation-insensitive restriction enzyme(Msp I) digests were used as target DNA and methylation-sensitiverestriction enzyme (Hpa II) digests as competitor DNA. Southernblot analysis indicated that 60 to 70% of the clones in thelibrary were derived from the methylated sites and overall enrichmentwas 200- to 1000-fold. IGCR was further applied to constructa library for the sites differentially methylated between brainand liver DNA. In the library, approximately 20% of the HpaII sites exhibited different degrees of methylation betweenthese tissues.     

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.     

Isolation and regional localization of DNA segments revealing polymorphic loci from human chromosome 13.

A recombinant DNA library enriched for portions of human chromosome 13 has been constructed from a hamster-human somatic cell hybrid that contained human chromosomes 13, 12, and 6p. A total of 733 phages were identified that contain human DNA inserts, and 46 single-copy subfragments have been derived and used as probes on Southern transfers of genomic DNA isolated from unrelated individuals. From this set, nine fragments revealing polymorphic loci (RFLP) in Msp I- or Taq I-digested DNA have been identified, of which three are polymorphic with both enzymes. Six of these probes have been shown to segregate concordantly with human chromosome 13 in a somatic cell hybrid mapping panel, and the RFLPs at these loci have been shown to behave as codominant Mendelian alleles. Additionally, hybridization to DNA isolated from cells containing various deletions of chromosome 13 has allowed regional localization. This recombinant DNA library will be useful in the study of retinoblastoma as well as in the study of the mechanisms responsible for abnormalities of this autosome.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Enrichment of specific genes from genomic DNA or from clone library DNA, using R-looping

We have developed a procedure for enriching DNA for specific sequences that is based on R-looping (Thomas et al., 1976). R-loops are formed with the DNA using mRNAs containing the sequence of interest and then isolated on poly(U)-sepharose via the poly(A) tail of the mRNA. Model experiments showed that plasmid DNA containing a cDNA copy of an immunoglobulin kappa chain mRNA could be selectively retrieved using this procedure. Approx. 5-10% of the kappa sequences in mouse embryo DNA could be recovered by R-looping, while non-specific binding of mouse DNA to the poly(U)-sepharose column was 0.03-0.04%. This represents a 100-200-fold enrichment of mouse genomic kappa sequences. We have also used the procedure to rapidly screen a mouse clone library for immunoglobulin heavy chain genes. DNA from the clone library was enriched 100-200-fold using immunoglobulin heavy chain mRNAs, and the enriched DNA repackaged in vitro to recover the phage.     

Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.     

A DNA polymorphism in close physical linkage with the proopiomelanocortin gene

Cellular DNAs from a panel of 20 unrelated individuals were screened for restriction fragment length polymorphisms (RFLP) with a DNA probe containing the first exon of the proopiomelanocortin gene (POMC), which has been assigned to chromosome 2p23-25. Digestion with the restriction endonuclease Sst 1 revealed a high frequency RFLP. The two alleles that were found are fragments of 10- and 15-kilobase (kb) length and are in Hardy-Weinberg equilibrium with frequencies of 72.6% and 27.4%, respectively. Informative families were tested for linkage between POMC/Sst 1 RFLP and other polymorphic markers of chromosome 2. Linkage was excluded to AcP-1 (2p23-25) at 15% recombination, which is still consistent with the chromosomal assignments for these genes. The close physical linkage (10 kb) of the polymorphic locus to the POMC gene makes this RFLP a suitable marker for future linkage studies involving the POMC gene.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

First domain encoding sequence mediates human class II beta-chain gene cross-hybridization

HLA class II molecules are surface heterodimers which are essential in the initiation of immune responses. The amount of polymorphism expressed by the different class II molecules is largely dependent on the polymorphic structure of their beta chains. Cross-hybridization between, class II beta genes frequently hampered restriction fragment length polymorphism analysis of donor genomic DNA. In this report we show that the cross-hybridization between human class II beta genes is mediated by a region of high homology, rich in C and G residues, between the first domain encoding sequences of DP, DQ, and DR genes. The removal of the DNA segment containing this region from the fragments used as labeled probes against the corresponding fragments of the genes at other loci or against endonuclease digested genomic DNA completely eliminated or drastically reduced the cross-hybridization. Also, the RFLP patterns generated with the shortened probes were more informative and much simpler to interpret than were these generated with probes made from the original genes.     

The use of random amplified polymorphic DNA markers in wheat

Summary An evaluation was made of the use of random amplified polymorphic DNA (RAPD) as a genetic marker system in wheat. Reproducible amplification products were obtained from varietal, homozygous single chromosome recombinant line and wheat/alien addition line genomic DNA with selected primers and rigorously optimized reaction conditions. Factors influencing the RAPD patterns are DNA concentration, Mg²⁺ concentration, polymerase concentration and denaturing temperature. In wheat, the non-homoeologous, non-dose responsive and dominant behaviour of RAPD products devalues their use as genetic markers for the construction of linkage maps, and the high probability that the amplified fragments derive from repetitive DNA limits their use as a source of conventional RFLP probes. However, RAPD markers will most certainly find many applications in the analysis of genotypes where single chromosomes or chromosome segments are to be manipulated.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones

 We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine (Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use. By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics. Received: 10 February 1998 / Accepted: 25 February 1998     

Identification of Molecular Marker for Septumless Bold Pod in Brassica campestris

Two cultivars of yellow sarson (Brassica campestris), B9 and NC1 have sharp phenotypic differences: a) pubescent or glabrous leaves, b) septumed pod with less number or septumless bold pod with higher number of seeds, and c) erect or drooping pods relative to stem axis. It is established that septum less pod is related to enhancement of seed yield and also related to high shattering resistance. The character septumed pod and pubescent leaf are controlled by single genes with complete dominance and are situated on the same linkage group, as evidenced by the study of F₁, F₂, F₃ and back cross population. To identify some DNA markers associated with septumless pod, firstly, Restriction Fragment Length Polymorphism (RFLP) between the parents were searched using 30 nonrepetitive clones picked from 89 partial genomic library as probes, secondly, Randomly Amplified Polymorphic DNA (RAPD) analysis was done by using 45 random decamer primers. RFLP analysis produced 182 discrete monomorphic bands i.e., they are unable to differentiate the two parents. In RAPD analysis, six primers produced 15 polymorphic fragments out of total 430 bands amplified by 45 primers. Among them A8-350, A10-250, A10-560 RAPD bands are expected to be linked with septumless bold pod and A3-720 with pub locus as evidenced from the bulked segregant analysis (8SA). These RAPD marker fragments of DNA were subsequently used as RFLP probes. A8-350 used as a probe revealed polymorphic bands in Eco RI digested parental DNA and also showed linkage both with septumless and glabrous loci in BSA. Approximate likelihood estimator of genetic distance between septumless locus and the marker is 1.67 cM as calculated through pooled sample mapping.     

A kinetic model for subtractive hybridization.

Nucleic acid sequences that differ in abundance between two populations (target sequences) can be cloned by multiple rounds of subtractive hybridization and amplification by PCR. These sequences can be cDNAs representing up-regulated mRNAs, or genomic DNAs from deletion mutants. We have derived an equation that describes the recovery of such sequences, and have used this to simulate the outcome of up to 10 rounds of subtractive hybridization and PCR amplification. When the model was tested by comparing its predictions with the published results from genomic and cDNA subtractions, the predictions of the model were generally in good agreement with the published data. We have modelled the outcomes of genomic subtractions, for a variety of genomes, and have used it to compare various strategies for enriching targets. The model predicts that for genomes of less than 5 x 10(8) bp, deletions of as small as 1 kbp should represent > 99% of the DNA after three to six rounds of hybridization (depending on the enrichment procedure). As genomes increase in size, the kinetics of hybridization become an important limiting factor. However, even for genomes as large as 3 x 10(9) bp, it should be possible to isolate deletions of 5 kbp using the appropriate conditions. These simulations suggest that such methods offer a realistic alternative to chromosome walking for identifying genomic deletions for which there are known phenotypes, thereby considerably reducing time and effort. For cDNA subtractive hybridization, the model predicts that after six rounds of hybridization, sequences that do not differ in abundance between the tester and driver populations (the background) will represent < 1% of the subtracted population, and even quite modestly upregulated cDNAs should be successfully enriched. Where several up-regulated cDNAs are present, the predicted final representation is dependent on both the initial abundance and the degree of up-regulation.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs

When a sex hormone-binding globulin (SHBG) cDNA was used to screen a human testicular cDNA library, three distinct cDNAs were isolated, one of which corresponds to the human SHBG cDNA sequence and probably represents testicular androgen-binding protein. The other two SHBG-related cDNAs each contain unique 5' regions that diverge from the SHBG cDNA sequence at the same position, and one of them (SHBGr-2) lacks a 208-base pair region within the SHBG cDNA. As a result, this cDNA could potentially encode for a truncated form of SHBG which lacks N-linked carbohydrates and part of the steroid-binding domain. Southern blots of human placental DNA and cloned genomic DNA fragments also indicate that SHBG and its related testicular cDNAs are the products of a single gene. Sequence analysis of the gene indicates that the complete coding region for the SHBG precursor is comprised of 8 exons, which are distributed over 3.2 kilobase (kb) of genomic DNA, and the unique 5' regions associated with the two SHBG-related testicular cDNAs were identified 1.9 kb upstream from the initiating codon for SHBG. In addition, the deletion within SHBGr-2 is due to the removal of exon 7, and an interesting feature of the gene is that differentially used exons are preceded by Alu repetitive DNA sequences. Although the relative abundance of the various SHBG-related mRNAs in the testis has not been established, Northern blot analysis indicates that they are similar in size (1.6 kb) to that of hepatic SHBG mRNA.     

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.