RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein–Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

Mosquito transferrin, an acute-phase protein that is up-regulated upon infection

When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3′ end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312–321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.

Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

Breaksite batch mapping, a rapid method for assay and identification of DNA breaksites in mammalian cells

DNA breaks occur during many processes in mammalian cells, including recombination, repair, mutagenesis and apoptosis. Here we report a simple and rapid method for assaying DNA breaks and identifying DNA breaksites. Breaksites are first tagged and amplified by ligation-mediated PCR (LM-PCR), using nested PCR primers to increase the specificity and sensitivity of amplification. Breaksites are then mapped by batch sequencing LM-PCR products. This allows easy identification of multiple breaksites per reaction without tedious fractionation of PCR products by gel electrophoresis or cloning. Breaksite batch mapping requires little starting material and can be used to identify either single- or double-strand breaks.

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ∼25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.

Differential display of intestinal mRNAs regulated by dietary zinc.

Regulation of gene expression by zinc is well established, especially through the metal response elements of the metallothionein genes; however, most other aspects of the functions of zinc in gene expression remain unknown. We have looked for intestinal mRNAs that are regulated by dietary zinc status. Using the reverse transcriptase-PCR method of mRNA differential display, we compared intestinal mRNA from rats that were maintained for 18 days in one of three dietary groups: zinc-deficient, zinc-adequate, and pair-fed zinc-adequate. At the end of this period, total RNA was prepared from the intestine and analyzed by mRNA differential display. Under these conditions, only differentially displayed cDNA bands that varied in the zinc-deficient group, relative to the zinc-adequate groups, were selected. Utilizing two anchored oligo-dT3' PCR primers and a total of 27 arbitrary decamers as 5' PCR primers, our results yielded 47 differentially displayed cDNA bands from intestinal RNA. Thirty were increased in zinc deficiency, and 17 were decreased. Nineteen bands were subcloned and sequenced. Eleven of these were detectable on Northern blots, of which four were confirmed as regulated. Three of these have homology to known genes: cholecystokinin, uroguanylin, and ubiquinone oxidoreductase. The fourth is a novel sequence as it has no significant homology in GenBank. The remainder of those cloned included novel sequences, as well as matches to reported expressed sequence tags, and functionally identified genes. Further characterization of the regulated sequences identified here will show whether they are primary or secondary effects of zinc deficiency.

Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a poly(A)+ tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3'-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

A tyrosine kinase profile of prostate carcinoma.

Tyrosine kinases play central roles in the growth and differentiation of normal and tumor cells. In this study, we have analyzed the general tyrosine kinase expression profile of a prostate carcinoma (PCA) xenograft, CWR22. We describe here an improved reverse transcriptase-PCR approach that permits identification of nearly 40 different kinases in a single screening; several of these kinases are newly cloned kinases and some are novel. According to this, there are 11 receptor kinases, 9 nonreceptor kinases, and at least 7 dual kinases expressed in the xenograft tissue. The receptor kinases include erbB2, erbB3, Ret, platelet-derived growth factor receptor, sky, nyk, eph, htk, sek (eph), ddr, and tkt. The nonreceptor kinases are lck, yes, abl, arg, JakI, tyk2, and etk/bmx. Most of the dual kinases are in the mitogen-activating protein (MAP) kinase-kinase (MKK) family, which includes MKK3, MKK4, MEK5, and a novel one. As a complementary approach, we also analyzed by specific reverse transcriptase-PCR primers the expression profile of erbB/epidermal growth factor receptor family receptors in a variety of PCA specimens, cell lines, and benign prostatic hyperplasia. We found that erbB1, -2, and -3 are often coexpressed in prostate tissues, but not in erbB4. The information established here should provide a base line to study the possible growth and oncogenic signals of PCA.

Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus.

The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment.

Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium.

Rat skeletal muscle selenoprotein W cDNA was isolated and sequenced. The isolation strategy involved design of degenerate PCR primers from reverse translation of a partial peptide sequence. A reverse transcription-coupled PCR product from rat muscle mRNA was used to screen a muscle cDNA library prepared from selenium-supplemented rats. The cDNA sequence confirmed the known protein primary sequence, including a selenocysteine residue encoded by TGA, and identified residues needed to complete the protein sequence. RNA folding algorithms predict a stem-loop structure in the 3' untranslated region of the selenoprotein W mRNA that resembles selenocysteine insertion sequence (SE-CIS) elements identified in other selenocysteine coding cDNAs. Dietary regulation of selenoprotein W mRNA was examined in rat muscle. Dietary selenium at 0.1 ppm as selenite increased muscle mRNA 4-fold relative to a selenium-deficient diet. Higher dietary selenium produced no further increase in mRNA levels.

Whole genome analysis: Experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR

The recent ability to sequence whole genomes allows ready access to all genetic material. The approaches outlined here allow automated analysis of sequence for the synthesis of optimal primers in an automated multiplex oligonucleotide synthesizer (AMOS). The efficiency is such that all ORFs for an organism can be amplified by PCR. The resulting amplicons can be used directly in the construction of DNA arrays or can be cloned for a large variety of functional analyses. These tools allow a replacement of single-gene analysis with a highly efficient whole-genome analysis.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.