PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping.

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

The HRAS1 minisatellite locus and risk of ovarian cancer.

Approximately 10% of ovarian cancers are due to mutations in highly penetrant inherited cancer susceptibility genes. The highly polymorphic HRAS1 minisatellite locus, located just downstream from the proto-oncogene H-ras-1 on chromosome 11p, consists of four common progenitor alleles and several dozen rare alleles, which apparently derive from mutations of the progenitors. Mutant alleles of this locus represent a major risk factor for cancers of the breast, colorectum, and bladder, and it was found that BRCAI mutation carriers with at least one rare HRAS1 allele have a greater risk of ovarian cancer than BRCA1 carriers with only common HRAS1 alleles. There are no conclusive studies of HRAS1 alleles in sporadic epithelial ovarian cancer. A case-control study of HRAS1 alleles was performed on DNA from 136 Caucasian patients with ovarian cancer and 108 cancer-free controls using conventional (Southern blot) and PCR-based methods to determine the frequency of rare HRAS1 alleles. Odds ratios (ORs) were estimated using unconditional logistic regression methods. A single degree of freedom test was used to assess the significance of linear trend across categories of increasing exposure. A statistically significant association between rare HRAS1 alleles and risk of ovarian cancer was observed [OR, 1.70; 95% confidence interval (CI), 1.03-2.80; P = 0.04]. Having only one rare allele was associated with a relative risk of 1.66 (95% CI, 0.91-3.01), whereas having two rare alleles increased the relative risk to 2.86 (95% CI, 0.75-10.94; trend P = 0.03). Analysis of HRAS1 allele types by the age of the case at diagnosis revealed that younger cases (<45 years) had a borderline statistically significant increased association with rare HRAS1 alleles compared to older cases (> or = 0 years; OR, 1.89; 95% CI, 0.90-3.98; P = 0.09). Rare HRAS1 alleles contribute to ovarian cancer predisposition in the general population. Thus, the HRAS1-variable number of tandem repeats locus may function as a modifier of ovarian cancer risk in both sporadic and hereditary ovarian cancer.

Instability of the EPM1 minisatellite.

Inherited mutations in the cystatin B gene ( CSTB ) are responsible for progressive myoclonus epilepsy type 1 (EPM1; MIM 254800). This autosomal recessive disease is characterized by variable progression to mental retardation, dementia and ataxia. The majority of EPM1 alleles identified to date contain expansions of a dodecamer repeat located upstream of the transcription start site of the CSTB gene. Normal alleles contain two or three copies of the repeat, whereas pathogenic alleles contain >40 repeats. We examined the meiotic stability of pathogenic, expanded EPM1 alleles from 17 EPM1 families by employing a fluorescence-based PCR-based genotyping assay capable of detecting single dodecamer repeat unit differences on an automated DNA sequencer. We followed 74 expanded allele transmissions to 30 affected individuals and 22 carriers. Thirty-five of 74 expanded allele transmissions demonstrated either contraction or expansion of the minisatellite, typically by a single repeat unit. Thus expanded alleles of the EPM1 minisatellite demonstrate a mutation rate of 47%, the highest yet observed for pathogenetic alleles of a human minisatellite.

Distinct mutation patterns of breast cancer-associated alleles of the HRAS1 minisatellite locus.

DNA sequence analysis of 130 alleles of the HRAS1 minisatellite has demonstrated that breast cancer-associated variants arise as a consequence of both replication errors and gene conversions. Unlike mutations at other variable number of tandem repeats (VNTRs), high-risk variants of the HRAS1 minisatellite do not demonstrate positional polarity. Instead, most mutations occur at three hotspots, with replication errors confined to one hotspot, gene conversions to a second and a mixed pattern of mutation at the third. DNA sequence analysis of 66 low-risk a1 alleles revealed no evidence for hypermutation. Therefore, while the HRAS1 minisatellite may serve as a reporter for a broad-based group of mutational mechanisms, these results are consistent with a direct pathogenetic contribution by high-risk alleles as the biological basis underlying cancer association of this VNTR.

Virion encapsidation of tRNA(3Lys)-ribozyme chimeric RNAs inhibits HIV infection.

Retroviruses require a specific host cellular tRNA primer for initiation of first-strand DNA synthesis. This primer is bound by viral proteins and copackaged into virions. We have exploited this property in the design and testing of an antiviral ribozyme fused to tRNA(3Lys), the primer used for lentiviral replication, including human immunodeficiency virus (HIV-1 and HIV-2). The chimera consists of tRNA(3Lys) covalently attached to a hammerhead ribozyme, which is targeted to the region immediately upstream of the primer binding site of the HIV-1 genome. The tRNA-ribozyme chimeric transcript is catalytically active in vitro and is efficiently bound by HIV reverse transcriptase with an affinity similar to that of tRNA(3Lys). We have expressed the chimeric RNAs from either the tRNA(3Lys) intragenic RNA polymerase III promoter or from a human U6 snRNA promoter. The U6 promoter results in up to 10-fold enhanced expression of the tRNA-ribozyme. Most importantly, the tRNA(3Lys)-ribozymes are encapsidated in HIV-1 virions such that they are effective in substantially reducing the level of infectious virus produced from cells cotransfected with HIV-1 proviral DNA. These results demonstrate the feasibility of using this novel strategy to reduce HIV infectivity and more generally indicate the potential power of using the retroviral primer tRNAs as tools for expressing and delivering ribozymes and other antiretroviral RNAs to the virion capsid.

An allelic variant at the ATM locus is implicated in breast cancer susceptibility.

We have tested a simple procedure, disease association by locus stratification, for identifying breast cancer patients with pathogenetic allelic variants at several candidate loci. The strategy was based on the assumption of epistatic interactions of the candidates. We analyzed 66 independent cases from sib pairs affected with breast cancer that had previously been collected during an investigation of pathogenetic-allele-sharing at the HRAS1 mini-satellite locus. An exon 24 polymorphism of ATM, substituting arginine for proline was associated with breast cancer in these cases with an overall odds ratio of 4.5 (95% confidence interval, 1.2-20.5, nominal p = 0.02, 2-tail Fisher exact test). In the presence of a rare HRAS1 allele, the odds ratio increased to 6.9 (95% CI, 1.2-38.3, p = 0.03). Thus, our procedure identified at least one allelic variant of ATM associated with breast cancer, and indicated that the ATM locus may interact with HRAS1.

A chimeric tRNA(Lys3)-ribozyme inhibits HIV replication following virion assembly.

Co-localization of ribozymes with their appropriate target is one method utilized to increase their effectiveness in vivo. Effective antiviral ribozymes will likely rely on mechanisms which direct the ribozyme to the genomic or subgenomic RNAs. Exploiting the fact that a specific host cellular tRNA primer is bound by viral proteins and co-packaged with viral genomes in newly synthesized virions, ribozymes were fused to the 3'-terminus of tRNA(Lys3) in an attempt to direct their activity to cleave the HIV-1 genome. This chimeric ribozyme is catalytically active in vitro, and is efficiently recognized and bound by HIV-1 reverse transcriptase with affinities similar to tRNA(Lys3). The intragenic RNA polymerase III promoter entity of the tRNA allows for high levels of expression of the tRNA-RBZ and the preferential localization of transcript within the cytoplasm in transfected cells. This ribozyme was effective in reducing the infectivity of a viral stock which was produced from transiently transfected cells bearing the chimeric gene. These results demonstrate the feasibility of using tRNAs as a means of co-localizing ribozymes with their viral genomic RNA targets. The possibility exists to fuse stable RNAs to ribozymes as a means of increasing their stability and localizing them to their appropriate target sites.

Isolation and functional analysis of a Kluyveromyces lactis RAP1 homologue.

Saccharomyces cerevisiae RAP1 (Sc RAP1) is an essential protein which interacts with diverse genetic loci within the cell. RAP1 binds site-specifically to the consensus sequence, 5'-AYCYRTRCAYYW (UASRPG, where R = A or G, W = A or T, Y = C or T). In Kluyveromyces lactis (Kl) ribosomal protein-encoding genes (rp) retain functional RAP1-binding elements, suggesting the presence of a RAP1-like factor. Kl extracts display an activity capable of specifically binding to rp fragments bearing UASRPG. We subsequently isolated the Kl RAP1-encoding gene by homology to a subfragment which encodes the N terminus of the DNA-binding domain of Sc RAP1. The predicted amino acid (aa) sequence of Kl RAP1 indicates it is smaller than Sc RAP1 (666 vs. 827 aa) with the N terminus being truncated. The DNA-binding domain is virtually identical between the two RAP1 proteins, while the RIF1 domain is moderately conserved. The region between these two domains and the N-termini are highly divergent. Two potential UASRPG were identified in the 5' flanking region, suggesting an autoregulatory role for RAP1. Despite the similarities between the two proteins, KI RAP1 is unable to complement Sc rap1ts mutants, suggesting that domains essential for function in Sc are absent from the Kl protein.

Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes.

Gene therapy for AIDS requires the identification of genes which effectively inhibit HIV-1 replication coupled to an efficient vector system for gene delivery and expression. Hammerhead ribozymes are RNA molecules capable of catalytic cleavage of complementary RNA molecules. Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave HIV RNA in the tat gene (TAT) or in a common exon for tat and rev (TR). The ribozymes were cloned into the LN (LTR-neomycin) retroviral vector plasmids and expressed as part of viral LTR-driven transcripts. The vectors were packaged as amphitropic virions and used to transduce human T-lymphocytes. Expression of the vector transcripts containing the ribozyme sequences was readily detected by Northern blot analysis of the transduced T cells. The T-lymphocytes expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication. In contrast, cells expressing mutant ribozymes, containing substitutions of a key nucleotide in the catalytic domain which cripples the cleavage activity of the ribozymes, supported replication of HIV-1, demonstrating that the functional ribozymes were cleaving the target RNAs. These studies demonstrate that retrovirally transduced ribozymes included in long, multifunctional transcripts, can inhibit HIV replication in human T-lymphocytes. The ribozyme and expression strategies described here should be useful for the gene therapy of AIDS by conferring resistance to HIV-1 replication on cells derived from transduced hematopoietic stem cells.

Altered response to growth rate changes in Kluyveromyces lactis versus Saccharomyces cerevisiae as demonstrated by heterologous expression of ribosomal protein 59 (CRY1)

We report the cloning, characterization and preliminary analysis of the regulation of the gene coding for ribosomal protein 59 (RP59) from the budding yeast Kluyveromyces lactis. The RP59 gene is present as a single copy, contains an intron within the amino terminal coding portion of the gene, and harbors conserved S. cerevisiae splicing signals. Sequence elements upstream of the transcriptional start site are homologous to UASRPG, known to regulate the transcription of numerous genes in S. cerevisiae via their interaction with the trans-activating factor RAP1. These elements are necessary for transcription of RP59 in both K.lactis and S.cerevisiae hosts. UASRPG in S.cerevisiae rp genes also modulate the transcription of rp RNA synthesis in response to a growth rate upshift. In K.lactis, the RP59 gene does not respond to growth rate upshift. Reciprocal expression of RP59 and CRY1 in heterologous hosts demonstrates that glucose upshift occurs in S.cerevisiae but not K.lactis. These results demonstrate that a factor or factors required for growth upshift are lacking in K.lactis, and provide further evidence that the UASRPG are sufficient signals for modulating this response.

Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.

Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene.

Plasmids carrying gene fusions between the yeast (Saccharomyces cerevisiae) actin gene and an initiation-defective Escherichia coli lacZ (beta-galactosidase) gene have been constructed. Expression of beta-galactosidase in such fusion plasmids depends on transcription of the actin gene, and is possible only after the RNA-splicing machinery has removed from the primary RNA transcript the 309-bp intervening sequence (IVS) interrupting the actin coding region. Mutants deleting the actin IVS were constructed via synthetic oligonucleotide-mediated in vitro mutagenesis of the actin-beta-galactosidase fusion plasmid. A 17-base synthetic oligonucleotide was used to generate a 309-bp deletion which precisely removed the actin IVS. A partial deletion mutant was also constructed in which 272-bp, starting at the 5' end of the actin IVS, and including the 5' splice junction signal, were deleted. Both the complete and partial IVS-deletion mutants were transformed into yeast hosts. However, the partial deletion resulted in a greater than 98% reduction in beta-galactosidase activity. The precise deletion of the actin IVS did not reduce the levels of beta-galactosidase activity as compared with the parental fusion plasmid containing the intact IVS.

Functional expression of a yeast ochre suppressor tRNA gene in Escherichia coli.

The yeast tRNATyr ochre suppressor SUP6 gene and a derivative of this gene in which the 14-bp intervening sequence has been deleted, SUP6 delta, have been examined for functional expression in Escherichia coli. The SUP6 delta, but not the SUP6, gene codes for a functional transfer RNA which has been shown to suppress both ochre and amber nonsense mutations in E. coli. Although the SUP6 delta fragment is contained within a 750-bp restriction fragment, isolated from Saccharomyces cerevisiae, and contains no encoded CCA, the primary transcript, which originates from an E. coli promoter, approx. 1000 bp upstream, is processed to a mature, functional transfer RNA. The pattern of suppression, i.e., suppression of both ochre and amber mutants, is characteristic of E. coli ochre suppressing tRNAs and is in contrast to the pattern observed in yeast, where only ochre mutations are suppressed. The SUP6 delta encoded tRNA, although coding for tRNATyr in yeast, is not charged solely with tyrosine in E. coli. The functional expression of this mutant eukaryotic transfer RNA gene in E. coli affords a unique opportunity for studies of expression of a gene coding for a stable RNA, in both a prokaryotic and an eukaryotic host.