Isolation of murine telomere-proximal sequences by affinity capture and PCR

We describe a method of selectively enriching for murine telomere-proximal sequences using affinity capture followed by PCR amplification. The telomeric fragments were selected from NotI-digested and lambda exonuclease-resected mouse genomic DNA by annealing to a biotinylated riboprobe containing multiple copies of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively retained on a matrix with covalently bound avidin. The captured DNA was then specifically released by ribonuclease action, and PCR amplification was performed using mouse repeat primers. The PCR products were cloned and used to screen a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones analyzed gave telomere-proximal hybridization signals, indicating an at least 500-fold enrichment for telomere-proximal sequences.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

Computerized polymorphic marker identification: experimental validation and a predicted human polymorphism catalog

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called POMPOUS (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by POMPOUS to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. POMPOUS provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use POMPOUS to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.     

Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man

Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster.     

CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface

DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes. We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome. This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells. These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage P1, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences. Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains). In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis.     

Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system

Synthetic oligonucleotide primers based on cDNA sequence were used to amplify the region spanning intron 2 of the alpha-globin gene of the bivalve mollusc Anadara trapezia. Amplification of this region from individual clams showed highly polymorphic patterns. The sequence of this intron was found to include a number of mono- [d(T)n and d(C)n], di- [d(CA)n and d(CT)n] and tetranucleotide d(CTGT)n repeats which were found to be polymorphic with respect to the types and numbers of repeats present. Two separate repeat-containing polymorphic regions were located near each end of this intron. The repeat at the 3' end consisted of an unusual example of a d(T)n polymorphism at the position of the polypyrimidine tract usually involved in intron splicing. Thirteen individual cloned intron 2 sequences, derived by PCR amplification from pooled genomic DNA, were sequenced without finding two identical sequences. All of the sequenced clones contained microsatellite sequences.     

Variation in severity of cardiac disease in Holt-Oram syndrome

Dinucleotide microsatellites are useful for gene mapping projects. Depending upon definition of conservation, published estimates of dinucleotide microsatellite conservation levels vary dramatically (30% to 100%). This study focused on well-characterized genes that contain microsatellites in the human genome. The objective was to examine the feasibility of developing microsatellite markers within genes on the basis of the assumption of microsatellite conservation across distantly related species. Eight genes (Gamma-actin, carcinoembryonic antigen, apolipoprotein A-II, cardiac beta myosin heavy chain, laminin B2 chain, MHC class I CD8 alpha chain, c-reactive protein, and retinoblastoma susceptibility protein) containing large dinucleotide repeat units (N > or = 15), complete genomic structure information, and homologous gene sequences in a second species were selected. Heterologous primers were designed from conserved exon sequences flanking a microsatellite motif. PCR products from bovine and porcine genomic DNA were tested for the presence of microsatellite sequences by Southern blot hybridization with biotin-labeled (CA)12 oligonucleotides. Fragments containing microsatellites were cloned and sequenced. Homology was verified by sequence comparisons between human and corresponding bovine or porcine fragments. Four of sixteen (25%) cross-amplified PCR products contained dinucleotide repetitive sequences with repeat unit lengths of 5 to 23. Two dinucleotide repetitive sequences showed microsatellite length polymorphism, and an additional sequence displayed single-strand conformational polymorphism. Results from this study suggest that exploitation of conserved microsatellite sequences is a useful approach for developing specific genetic markers for comparative mapping purposes.     

Evaluation of brain tumor by 99mTc-MIBI: comparison study with 201Tl and predictivity of therapeutic effect

Spider silks are highly repetitive proteins, characterized by regions of polyalanine and glycine-rich repeating units. We have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One sequence (1726 bp) was from a cloned cDNA, and the other (1951 bp) was from PCR of genomic DNA. When these sequences are compared with each other and the previously published Spidroin 1 sequence, there are differences due to sequence rearrangements, as well as single base substitutions. These variations are similar to those that have been reported from other highly repetitive genes, and probably represent the results of unequal cross-overs. We have also obtained 708 bp of sequence from pCR of genomic DNA from Araneus biocentenarius. This sequence shows considerable similarity to a dragline sequence (ADF-3) from A. diadematus, as well as Spidroin 2 (NCF-2) from N. clavipes. Minor but consistent differences in the repeating unit sequence between A. bicentenarius and A. diadematus suggest that concerted evolution or gene conversion processes are acting to maintain similarity among repeat units within a single gene.     

Laser Doppler flowmetry: a clinical test of pulpal vitality

Norway spruce (Picea abies) genomic libraries were screened for presence of dinucleotide AC/GT and AG/CT microsatellites (or simple sequence repeats). On average, one (AG)n microsatellite every 194 kb and one (AC)n microsatellite every 406 kb were found. Forty-six positive clones were sequenced and primers flanking 24 AG microsatellites and 12 AC microsatellites diesigned. Only seven (20%) of them produced the expected single-locus polymorphic pattern when used to amplify Norway spruce DNAs. The other primer pairs gave either multiple bands or bad amplification, or a single monomorphic fragment. Such a small proportion of successful primer pairs was attributed to the high level of complexity of the Norway spruce genome. Dot blot analysis of the clones showed that many of them contained repetitive DNA and that those giving the single-locus polymorphic patterns usually corresponded to single-copy sequences. A family of repetitive DNA that contained AG repeats was identified and was present in about 40,000 copies per haploid genome. Simple Mendelian inheritance was observed for all the polymorphisms tested. The average number of alleles was 13, ranging from 6 to 22, and the expected heterozygosity was 0.79 when seven microsatellites were used to genotype a panel of 18 trees representing different populations. Compared with isozymes, microsatellites are about five times more informative and could provide an extremely valuable source of markers for genome mapping and genetic diversity studies.     

Microsatellite enrichment in organisms with large genomes (Allium cepa L.)

To exploit the polymorphism of repeat numbers in short tandem repeat (STR) sequences (microsatellites) as molecular markers, STRs must be isolated and PCR primers must be developed in flanking sequences. In species with large genomes such as Allium cepa L. (onion and shallot), an efficient selection procedure for genomic fragments containing STRs is a crucial step. Here we describe a nonradioactive method for microsatellite isolation based on affinity capture of single-stranded restriction fragments annealed to biotinylated microsatellite oligonucleotides (CA)10, (GAA)8 and (AAC)8 followed by adapter-mediated genomic PCR. Cloning of the products in E. coli and plasmid sequencing revealed more than 60% positive clones. Primers were designed in STR-flanking regions, and one or two bands were amplified in 13 diploid onion and five shallot accessions. Allelism of the bands was confirmed by product sequencing.     

Class II HLA antigens in patients with multiple sclerosis from Warsaw and Gdansk regions]

We have identified a novel 399 bp repetitive DNA element (which we designate beta) 9bp upstream of a seryl-tRNA(CAG) gene in the genome of Candida albicans. There are two copies of the seryl-tRNA(CAG) gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNA(CAG) gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNA(Asp), tRNA(Ala) and tRNA(Ile). The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae. We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes.     

Isolation and characterization of a satellite DNA family in the Saccharum complex

EaCIR1, a 371-bp Erianthus-specific satellite DNA sequence, was cloned from TaqI restricted genomic DNA after agarose-gel electrophoresis. This sequence has 77% homology with a 365-bp satellite of Helictotrichon convolutum and 72% homology with a 353-bp tandem repeat sequence from Oryza sativa. PCR primers defined in the conserved regions of these repetitive sequences were used to isolate other satellite DNAs in different representatives of the Saccharum complex: SoCIR1 in Saccharum officinarum, SrCIR1 in Saccharum robustum, SsCIR1 and SsCIR2 in Saccharum spontaneum, and MsCIR1 in Miscanthus sinensis. EaCIR1 and SoCIR1 were localized to subtelomeric regions of the chromosomes by fluorescence in situ hybridization. Southern hybridization experiments, using two representatives of this repeat sequence family as probes, illustrated contrasting species-specificity and demonstrated the existence of similar repetitive elements in sorghum and maize.     

Identification of 12p as a region of frequent deletion in advanced prostate cancer

The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.     

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.     

Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid

We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.     

Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.     

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.