Site-specific Primers Research Articles

Improved detection of Mycobacterium tuberculosis using two independent PCR targets in a tertiary care centre in South India

Tuberculosis (TB) causes significant morbidity and mortality worldwide as one of the leading infectious diseases. In India, more than 1.8 million new cases occur every year. Rapid and accurate diagnosis of TB would improve patient care and limit its transmission. This study aimed to evaluate a dual target polymerase chain reaction (PCR) diagnostic assay to detect Mycobacterium tuberculosis from pulmonary and extra-pulmonary samples at a tertiary care centre in South India. Samples were collected from patients with a low index of suspicion of TB. Acid-fast smears were performed by Auramine O fluorescent microscopy and PCR was performed by using two site-specific primer pairs targeting IS6110 by nested PCR and TRC4 by conventional PCR. Amplified products for IS6110 and/or TRC4 were indicative of M. tuberculosis. Among 114 (19 pulmonary and 95 extra-pulmonary) samples tested by PCR assay, 12 (11%) were positive for both IS6110 and TRC4, of which 11 (10%) were non-respiratory and one was (1%) respiratory in origin. PCR for TRC4 alone was positive for eight (7%) non-respiratory and two (2%) respiratory samples, while IS6110 alone tested positive for six (5%) non-respiratory samples and one (1%) respiratory sample. Of a total of 29 PCR positive samples, 17 (15 %) were acid-fast smear positive. Although the target site of IS6110 is specific for M. tuberculosis, some strains from South India may lack this region. Therefore, the use of an additional target site (TRC4) is required for improved detection of M. tuberculosis.

Translocation-Capture Sequencing Reveals the Extent and Nature of Chromosomal Rearrangements in B Lymphocytes

Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.

Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

BackgroundGroup 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species.ResultsIntron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE.ConclusionThe three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

Abstract 2959: Characterization and function of unique T-cell factor-4 splicing isoforms that exhibit tumor repressive phenotypes in hepatocellular carcinoma

Abstract Background and Aims: The canonical Wnt signaling is frequently activated in hepatocellular carcinoma (HCC). Although β-catenin is the principle effector protein of Wnt signaling, the success of relaying signals to the nucleus depends on the T-cell factor (TCF) family of transcription factors that bind to promoters of Wnt-responsive target genes. The human TCF-4 gene (TCF7L2) is composed of 17 exons with multiple alternative splicing sites. In spite of theoretical possibility of constructing more than 500 isoforms, only two TCF-4 splice variants have been identified so far. Therefore, the objective of this study is to identify different TCF-4 isoforms and explore their putative role in hepatic oncogenesis. Methods: The TCF-4 isoforms were detected by RT-PCR. Their expression was confirmed by using RT-PCR with splice site-specific primer pairs in HCC cell lines, HCC tumor and peritumor tissues, and normal liver. To examine their functional properties, mammalian expression plasmids encoding each isoform were prepared and protein products were verified by Western blotting. The TCF transcriptional activity, cell proliferation, migration and transformation were measured to evaluate their biological functions. Results: We identified and subsequently cloned 14 different TCF-4 splicing variants derived from HCC cell lines. Two previously known forms TCF-4B (short) and TCF-4E (long) were found among the 14 isoforms. The 12 new isotypes were named in alphabetical order according to increasing size of their protein products. Out of the 14 TCF-4 isoforms, we have detected 8 short forms (A-I except for E), 5 long forms (J-M plus E), and a novel isoform designated as TCF-4X. The structure of TCF-4X included an extra exon, labeled as exon 5a, between exons 5 and 6 indicating that TCF7L2 may have 18th exon in the genome. Functional analysis of these isotypes revealed several distinctive phenotypes. TCF-4B showed the highest TCF transcriptional activity and enhanced cell growth rate, but lower cell migration and transforming ability compared to control. TCF-4J exhibited lower transcriptional activity but the highest cell proliferation, migration and colony formation rate. In contrast, TCF-4K displayed the lowest TCF transcriptional activity, inhibited cell proliferation, and eliminated colony-forming ability. In addition, their expression profile by RT-PCR indicated that TCF-4J was highly expressed in HCC tumors compared to peritumor and normal liver, while TCF-4K was barely detected in HCC cell lines and tumor tissues. Conclusions: Our study demonstrates that at least 14 different TCF-4 isoforms are expressed in HCC and their function is distinguishable based on the structure of the splicing sites. Whereas the TCF-4J isoform produced the most characteristic features of the malignant phenotype, TCF-4K exhibited tumor repressive traits. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2959.

Transgene Copy Number and Integration Site Analysis in Transgenic Pig*

Transgene copy number and integration site were checked in transgenic pigs produced by somatic cell nuclear transfer (SCNT),moreover,Junction PCR was employed to confirm the integration site and analyze zygosity. The results showed that:absolute quantitative PCR could calculate transgene copy number efficiently. The parameters of the standard curve was:log2N=-0.935 4△Ct+3.411 6 (R2=0.997 4,P 0.001),and copy number were 30.85 ±1.77,18.87 ±1.34,respectively,in two transgenic pigs; transgene integration site was successfully cloned by TAIL-PCR,and 25 bands were obtained. Three integration sites,named TgInS1 (1 440 bp),TgInS2 (1 263 bp) and TgInS3 (1 861 bp),were detected by BLAST; Junction PCR combining with integration site and transgene specific primers was performed and specific bands confirmed the integration site; Junction PCR combining with 5' and 3' integration site and transgene specific primers was performed to analyze integration site zygosity. Bands amplified by 5' and 3' integration site specific primers just as WT control were obtained to determine the heterozygosity of integration site. Absolute quantitative PCR,and TAIL-PCR were established to check transgene copy number and integration site,and it has laid the foundation to study the inheritance and expression stability of transgene in transgenic animals.

Transgene Copy Number and Integration Site Analysis in Transgenic Pig*

Transgene copy number and integration site were checked in transgenic pigs produced by somatic cell nuclear transfer (SCNT),moreover,Junction PCR was employed to confirm the integration site and analyze zygosity. The results showed that:absolute quantitative PCR could calculate transgene copy number efficiently. The parameters of the standard curve was:log2N=-0.935 4△Ct+3.411 6 (R2=0.997 4,P 0.001),and copy number were 30.85 ±1.77,18.87 ±1.34,respectively,in two transgenic pigs; transgene integration site was successfully cloned by TAIL-PCR,and 25 bands were obtained. Three integration sites,named TgInS1 (1 440 bp),TgInS2 (1 263 bp) and TgInS3 (1 861 bp),were detected by BLAST; Junction PCR combining with integration site and transgene specific primers was performed and specific bands confirmed the integration site; Junction PCR combining with 5' and 3' integration site and transgene specific primers was performed to analyze integration site zygosity. Bands amplified by 5' and 3' integration site specific primers just as WT control were obtained to determine the heterozygosity of integration site. Absolute quantitative PCR,and TAIL-PCR were established to check transgene copy number and integration site,and it has laid the foundation to study the inheritance and expression stability of transgene in transgenic animals.

Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease

In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA(1350-1784), OmpB(801-1269,) and OmpB(1227-1634) regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii. For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA(1350-1784) (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB(801-1269) and OmpB(1227-1634) were 90% and 95%, respectively. The specificities of the OmpB(801-1269) and the OmpB(1227-1634) were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii, and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.

Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection.

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV (Blood . 2002; 99:698–701). Half of the donor cells were genetically modified with a Moloney murine leukemia virus (MoMLV) based HIV resistance vector containing a transdominant negative mutant Rev (TdRev) (2.58×10e8 cells) or a control vector MoMLV based vector encoding GP91phox (4.04×10e8 cells). Here we report an assessment of retroviral integration sites recovered out to 3 years post-transplantation.We identified 213 unique retroviral integration sites (RISs) from the patient's peripheral blood samples myeloid and lymphoid cells from 1 to 36 months after reinfusion of genetically modified CD34+ cells by linear amplification-mediated PCR (LAM-PCR). While overall vector integration patterns were similar to that previously reported, only 3.75% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). This low percentage of overlap offers further evidence that the early phase of hematopoiesis after transplantation derives primarily from short-term repopulating cells. Additionally, we identified 14 common integration sites (CISs). Interestingly, common integration sites were enriched among late samples; 14.9% of early RISs were CISs vs. 36.8% late.A total of 3 RISs were found near or within known oncogenes, but 2 (Integrin alpha 9 [ITGA9] and ADP-ribosylation factor-like 11 [ARL11]) were limited to early time points. An integration site near the MDS1 gene was detected in a late follow-up sample by LAM-PCR. We confirmed the integration site near the MDS1 gene by PCR with integration site-specific primers amplifying the region between the 3'-LTR of the provirus and the MDS1 locus. The MDS1 integration was not detected in early, but became detectable at all time points from 6 months to 3 years post transplant from both lymphoid and myeloid populations. Q-PCR using an integration specific Taqman probe was utilized to assess the level of clonal contribution to hematopoiesis from the clone containing the MDS1 RIS. The overall contribution of the MDS1 integrated clone remained stable during followup. Given an overall gene marking level of 0.001-0.01% with an MDS1 marking level estimated at 0.00001% in the follow up samples, the frequency of the MDS1 integrated clone is predicted to be 1/1000 marked LT-HSCs. We infused an estimated 1324 transduced LT-HSCs based upon cell dose, transduction efficiency and an estimated LT-HSC frequency of 5 per 10e3 CD34+ cells. The single integration in MDS1 in the context of non-LT-HSC limited hematopoiesis may thus account for the stability observed over time.In summary, the pattern of contribution by genetically modified cells is distinct between the early and late phase post transplantation and emphasizes the importance of long-term studies to assess the risk of integrating vectors. Additionally, the enrichment for CISs in the late phase supports the concept that integrations in the LT-HSCs favors genes that may be involved in “stemness”. Furthermore, integrations in or near putative oncogenes are likely insufficient alone as a cause of oncogenesis. Finally, LT-HSC dose may be an important determinant of the risk of integrating vectors in the context of HSC gene transfer.

Retrieval of entire genes from environmental DNA by inverse PCR with pre‐amplification of target genes using primers containing locked nucleic acids

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity

When transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method-TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites. By way of example, using this approach, three separate integration sites were found (on chromosomes 8, 15 and 17) in one transgenic founder. As the sites on chromosomes 8 and 15 failed to segregate in any subsequent progeny, whole chromosome paints were done to determine if translocations involving chromosomes 8 and 15 occurred at the time of transgene integration. Whole chromosome painting could not detect translocations, suggesting that the rearrangements likely involve only small stretches of chromosomes. Site-specific primers were used to identify the progeny carrying only one integration site; these mice were then used as sub-founders for subsequent breedings. Integration site specific primers were used to distinguish homozygous progeny from heterozygotes. TAIL-PCR thus provides an easy and reliable way to (1) identify multiple integration sites in transgenic founders, (2) select breeders with one integration site, and (3) determine zygosity in subsequent progeny. Use of this strategy may also be considered to map integration sites in situations of unexpected phenotype or embryonic lethality while creating new transgenic mice.

Differentiation of Highly Conserved Host and Transgene mRNA in HO-1 Gene Therapy.

Background: Heme oxygenase-1 (HO-1) is highly inducible in vitro and in vivo and appears to confer protection to cells and tissues from oxidative stress and inflammation. Marked oxidative stress and vascular inflammation are hallmarks of sickle cell disease (SCD). We have previously shown that enhanced expression of HO-1 inhibits vascular inflammation and hypoxia-induced vaso-occlusion in murine models of SCD. To further these observations, we constructed transgene forms of rat HO-1 for gene therapy in murine models of SCD. The high degree of interspecies sequence conservation makes epitope choices for primary antibody and nucleic acid sequence available for probes or primers problematic for differentiation of endogenous and transgenic HO-1. There are only 16 differences in the 289 amino acids between murine and rat HO-1. At the genetic level, murine and rat HO-1 are 93% identical. We utilized interspecies RNA sequence differences to accurately differentiate endogenous murine HO-1 from rat transgenic HO-1.Methods: Two pairs of RT-PCR primers were designed from areas of interspecies identity. Both primer pairs generated cDNAs with 10 base pair differences between species. In addition, each flanked an interspecies single nucleotide difference, which represents a unique restriction site in the rat, but not the murine cDNA. The GGGCCC sequence in rat is an ApaI restriction endonuclease (RE) site whereas the murine GGGCCT is not. The second primer pair created an RE site through site specific primer induced restriction fragment length polymorphism (SSPI RFLP). Rat sequence at bp 375 is TCCTGA; murine is CCCTGA; and RE BspEI recognizes TCCGGA. Our primer incorporates a mismatch in its third position, modifying the PCR product to TCCGGA and CCCGGA, respectively. This rat cDNA can be digested with either ApaI or BspEI. PCR amplification of HO-1 was performed on quantified, DNAseI treated, ApaI digested total RNA with either primer pair in a 25ul one tube system that included RNase inhibitor.Results: The quantification of total HO-1 RNA relative contribution from each species was performed by densitometry via Molecular Analyst™ Program on 2% agarose gel electrophoresis separation of restriction enzyme digested total PCR product. Murine PCR products were 200 bp as they do not contain either restriction site. ApaI and BspEI digestion of rat PCR products produced bands at 130 bp and 70 bp or 175 bp and 25 bp, respectively. Control experiments utilizing liver or spleen total RNA from both species demonstrated that mixed samples produce digested cDNA that reflects their relative proportion by species and that the reaction is reliable in dilution series.Conclusions: RFLP and SSPI RFLP can be used to differentiate highly conserved transcripts of native HO-1 RNA from transgene HO-1 RNA. RFLP and SSPI-RFLP can be applied to discriminate native inducible transcripts from transgenes in gene therapy studies, when activity, Western blot and immunofluorescence are not viable options. We are using this novel methodology in our ongoing studies using rat transgenes in murine models of SCD.

In planta ORFeome analysis by large‐scale over‐expression of GATEWAY®‐compatible cDNA clones: screening of ERF transcription factors involved in abiotic stress defense

Genomic approaches have generated large Arabidopsis thaliana open reading frame (ORF) collections. However, tools are required to functionally characterize this ORFeome. Here we describe a batch procedure to simultaneously recombine a population of GATEWAY-tagged full-length cDNAs into a plant expression vector. A pool of agrobacteria carrying these constructs has been used in flower-dip transformation experiments to obtain a collection of transgenic lines that over-express HA-tagged ORFs. This AtTORF-Ex library can be used in various screening approaches to identify particular gene family members involved in plant development or stress responses. The feasibility of the approach was studied using a near-complete collection of the Arabidopsis ethylene response factor (ERF) transcription factor (TF) family. Quality control performed at each step of the procedure revealed that the complexity of the population is maintained, and that almost all members of the ORF collection are covered by the plant library. The frequency of multiple transformation events has been determined as approximately 4%. Significant transgene expression was detected at the RNA and protein level in more than 60% and 30% of the transgenic plants, respectively. Striking phenotypic alterations were observed in approximately 4% of the plants. Many ERF TFs have been shown to participate in plant stress responses. As a proof of principle, the AtTORF-Ex library has been used in a selection procedure to isolate TFs involved in enhanced abiotic stress tolerance. The corresponding TF gene can be easily polymerase chain reaction-amplified using GATEWAY att site-specific primers. In summary, we describe here a method that can be generally applied for functional analysis of ORFeomes in planta.

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) II. Extractives Content

Abstract We describe the development of a SCAR-marker linked to low extractives content of Norway Spruce (Picea abies L [Karst.]) derived from AFLPs. In these analyses 57 different primer enzyme combinations were used in a bulked segregant analysis approach comparing individuals with high and low extractives content. A total of 14 polymorphic AFLP markers were detected between the pools. Five markers were selected for further analyses to verify their linkage to extractives content based on individuals used for pool constitution. One AFLP marker, found to be significant linked to low extractives content was converted into a SCAR marker for further validation. For this marker, a monomorphic band was obtained by using sets of nested primers or restriction site specific primers (RSS) which include the AFLP-restriction recognition site. The separation of the marker from unlinked size homologous marker-alleles was realized by a SSCP-approach. Validation of the marker on different full-sib families confirmed the usability to separate the classes for low and high extractives content of Picea abies.

Convergence of Cell Cycle Regulation and Growth Factor Signals on GRASP65

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

234. Polyclonal Insertion Sites in Two Rhesus Macaques after Non-Myeloablative Transplantation with MFGS-gp91phox Transduced Autologous CD34+PBPC

Mutagenesis activation of the LMO-2 gene was observed to be associated with the development of lymphoid leukemia several years after two infants with X-SCID had their immune systems restored by gene therapy with a murine onco-retrovirus vector encoding the human IL2 receptor common gamma chain. Just recently, a third infant treated with the same vector was reported to also have developed T cell leukemia, however, insertion site analysis is pending in this case. These observations highlight the importance of the conductance of large animal marking studies which follow the insertional pattern of vectors planned for human treatments. We have recently reported that a high titer RD114 pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the oxidase defect in CD34+ cells from patients with X-linked chronic granulomatous disease in the xenotransplant NOD/SCID mouse model. In anticipation of considering clinical use of this vector, we transduced G-CSF-FLT3L fusion protein mobilized peripheral blood CD34+ cells (CD34+ PBPC) of two healthy rhesus macaques with the RD114-MFGS-gp91phox. Transduced CD34+PBPC were transplanted on culture day 4 into two non-myeloablative (2|[times]|300 Rads) irradiated animals. The in vivo marking decreased over the first months and stabilized at about 8 months post transplant. Marking for animal J976 and D406 at 15 and 19 months, respectively is 1.3% and 0.3% in B lymphocytes, 0.3% and 0.3% in neutrophils and 0.7% and 0.4% in T lymphocytes at which time the animals remained healthy. Vector insertion analysis was performed using LAM-PCR and gel electrophoresis. Analyses of cloned and sequenced integration sites revealed 44 integrations sites in monkey J976, and 33 integration sites in monkey D406 with no predominant clone seen to emerge over time. 27% (J976) and 12% (D406) of the insertion sites were identified as coding regions (intron or exon). Nested PCR with insertion site specific primers enabled recovery of identified sites from different cell lineages at different time points. In summary, we detected a high number of integration sites in two rhesus macaques transplanted with gene-modified CD34+ cells despite low marking due to non-myeloablative conditioning.

Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population1

A mutation (C677T) in the gene, MTHFR, is known to increase susceptibility to various multifactorial disorders. In order to assess this single nucleotide polymorphism (SNP) as risk factor for idiopathic male infertility, a case-control study was done on an Indian population. DNA from 151 cases of non-obstruction, idiopathic oligo-/azoospermia and 200 fertile males (controls) was polymerase chain reaction amplified using site-specific primers, and analysed for the mutation following HinfI-digestion. Our results show a significantly increased frequency of CT heterozygotes among infertile patients (p value <0.04). More importantly, while there were no T homozygotes in the control population, six of 151 infertile cases were T homozygous. Considering that T allele occurs in very low frequency in the control population, 677T is clearly a risk factor for infertility in the Indian population. We contend that the same could also be true for African and Southeast Asian populations where the frequency of 677T is very low. The lack of similar association in western populations could be because of the overall dietary enrichment of folates, which could nullify or minimize the effect of this polymorphism.

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) I. Wood Density

Abstract The identification of AFLP markers and their subsequent conversion to SCAR-markers linked to wood density of Norway Spruce (Picea abies L [Karst.]) is described for the first time. In AFLP-analyses, 102 different primer enzyme combinations were screened in a bulked segregant approach comparing individuals with high and low wood density. A total of 107 polymorphic AFLP fragments were obtained between the DNA-pools. Twenty-three markers were selected for further analyses to verify their linkage to wood density based on individuals used for pool constitution and additional unrelated clonal material. For 15 markers, a significant linkage to wood density was confirmed by a two-sided Fisher’s-exact test. Four markers were converted into SCAR markers and validated for plant material assayed for wood density by X-ray microdensitometry. For each marker a monomorphic band was obtained using sets of nested primers or restriction site-specific primers (RSS), which include the AFLP-restriction recognition sites. For two markers that are linked to high wood density, a separation from unlinked size homologous marker-alleles was realized by a PCR-restriction approach. Validation of these markers in different full-sib families confirmed their usability to separate the classes for low and high wood density of Picea abies.

Quantitation of mRNA levels of steroid 5α-reductase isozymes in the rat brain by “one-step” RT-PCR and capillary electrophoresis

The enzyme 5α-reductase (5α-R) is present in many mammalian tissues, including the brain. The physiological importance of 5α-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone to its 5α-reduced derivative, precursors of allopregnanolone, potent allosteric modulator of the γ-aminobutyric acid receptor (GABA A-R). 5α-R occurs as two isoforms, 5α-R type 1 (5α-R1) and 5α-R type 2 (5α-R2). We present an accurate, rapid, and modestly labor-intensive method to precisely quantitate 5α-R mRNA species in the cerebral cortex of the rat. This approach combines the high specificity of “one-step” reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were co-amplified in a single reaction in which the same primers are used. The method presented in this paper enables a more efficient quantitative determination of 5α-R mRNA isozymes, and may lead to a better understanding of the role of 5α-R isozymes in the physiology of the central nervous system.