PCR Bands Research Articles

Use of tall fescue EST-SSR markers in phylogenetic analysis of cool-season forage grasses

Microsatellites or simple sequence repeats (SSRs) are highly useful molecular markers for plant improvement. Expressed sequence tag (EST)-SSR markers have a higher rate of transferability across species than genomic SSR markers and are thus well suited for application in cross-species phylogenetic studies. Our objectives were to examine the amplification of tall fescue EST-SSR markers in 12 grass species representing 8 genera of 4 tribes from 2 subfamilies of Poaceae and the applicability of these markers for phylogenetic analysis of grass species. About 43% of the 145 EST-SSR primer pairs produced PCR bands in all 12 grass species and had high levels of polymorphism in all forage grasses studied. Thus, these markers will be useful in a variety of forage grass species, including the ones tested in this study. SSR marker data were useful in grouping genotypes within each species. Lolium temulentum, a potential model species for cool-season forage grasses, showed a close relation with the major Festuca-Lolium species in the study. Tall wheat grass was found to be closely related to hexaploid wheat, thereby confirming the known taxonomic relations between these species. While clustering of closely related species was found, the effectiveness of such data in evaluating distantly related species needs further investigations. The phylogenetic trees based on DNA sequences of selected SSR bands were in agreement with the phylogenetic relations based on length polymorphism of SSRs markers. Tall fescue EST-SSR markers depicted phylogenetic relations among a wide range of cool-season forage grass species and thus are an important resource for researchers working with such grass species.

ASePCR: alternative splicing electronic RT-PCR in multiple tissues and organs.

RT–PCR is one of the most powerful and direct methods to detect transcript variants due to alternative splicing (AS) that increase transcript diversity significantly in vertebrates. ASePCR is an efficient web-based application that emulates RT–PCR in various tissues. It estimates the amplicon size for a given primer pair based on the transcript models identified by the reverse e-PCR program of the NCBI. The tissue specificity of each PCR band is deduced from the tissue information of expressed sequence tag (EST) sequences compatible with each transcript structure. The output page shows PCR bands like a gel electrophoresis in various tissues. Each band in the output picture represents a putative isoform that could happen in a tissue-specific manner. It also shows the EST alignment and tissue information in the genome browser. Furthermore, the user can compare the AS patterns of orthologous genes in other species. The ASePCR, available at , supports the transcriptome models of the RefSeq, Ensembl, ECgene and AceView for human, mouse, rat and chicken genomes. It will be a valuable web resource to explore the transcriptome diversity associated with different tissues and organs in multiple species.

Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil

We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp. niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1 ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.

Assessing genetic diversity of some Anthurium andraeanum Hort. cut-flower cultivars using RAPD Markers

Randomly amplified polymorphic DNA (RAPD) markers fingerprinting were used to assess the level of genetic variations among 24 cut-flower Anthurium andraeanum Hort. cultivars. Eight decamer primers produced a total of 98 reproducible PCR bands that were used to calculate the Nei and Li’s genetic distance (GDNL) coefficients amongst the cultivars. GDNL values ranged from 0.018 to 0.163 with an average of 0.09 (representing an average genetic similarity of 91.34%). This significantly low average genetic distance among the various cultivars indicated that genetic variation among the cultivars was low. A dendrogram, produced using unweighted pair group method using arithmetic averages (UPGMA), grouped the cultivars into four main clusters. Cultivar ‘Antartica’ was genetically distinct from all the others. ‘Midori’ and ‘Bourgogne’ together formed a cluster whereas the remaining 21 cultivars grouped into two clusters and were closely related to each other. Clusters did not relate to cultivar provenance or origin and were independent of floral colour and spathe category. Finding correlations between these morphological traits to RAPD markers would necessitate extensive primer screening. Nevertheless, RAPD markers fingerprinting allowed a rapid assessment of the level of genetic variation that would otherwise be difficult to evaluate using the limited number of morphological markers present among these closely related anthurium cultivars.

Identification and Partial Characterization of the Nonribosomal Peptide Synthetase Gene Responsible for Cereulide Production in Emetic Bacillus cereus

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)(3), cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants. The valine-activating module was highly conserved when sequences from nine emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B. cereus strains.

Gene Expression of Heme Oxygenase-1: Relationship to Fibrogenic and Apoptotic Factors in Murine Schistosomiasis

Schistosomiasis is characterized by a unique form of periportal fibrosis and not cirrhosis. Heme oxygenase (HO-1) is a stress inducible molecule that could modulate the fibrogenic process in the murine model of schistosoma mansoni. In this study, the extent of heme oxygenase-1 gene expression was evaluated by RT-PCR in liver tissues of thirty mice infested with schistosoma mansoni after the 2nd, 4th, 6th, 8th and 10th weeks of infestation. Furthermore, the gene expression of some fibrogenic and antifibrogenic factors [pro α2 (I) collagen, laminin β1, transforming growth factor β (TGF-β), platelet derived growth factor (PDGF) and hepatocyte growth factor (HGF)], apoptotic factors (Fas L) and the transcriptional factor nuclear factor-κB (NF-κB) were also evaluated in liver tissues of the same animals by RT-PCR techniques. The PCR bands densities were semi-quantitated using Biometra gel documentation system. The present study showed that the gene expression of HO-1 coincided with the expression of NF-κB. Both were induced from the 2nd week post infestation and their PCR band density remained high till the 8th week with a slight decline at the 10th week. The gene expression of HO-1 paralleled the levels of HO activity. On the other hand, increase in gene expression of HGF and Fas L was detected only in the 2nd and the 4th weeks post infestation. This may suggest a down regulation of Fas L and HGF by the sustained increase in gene expression of HO-1 and/or NF-κB. Meanwhile, there is increase in the gene expression of the profibrogenic cytokines PDGF and TGF-β, starting in the 4th week (PDGF) and 6th week (TGF-β). They were however expressed at low levels at the 2nd and 4th weeks, respectively. Thus a direct effect of HO-1 on the expression of these cytokines cannot be sharply delineated. However, the expression of these cytokines preceded and then paralleled pro α2 (1) collagen gene expression, indicating their role in the fibrogenic process. Laminin gene expression increased from the 6th week and remained highly expressed till the 10th weeks. Thus the similar patterns of gene expression of HO-1 and NF-κB at the selected stages in murine schistosomiasis and the accompanying down regulation of the Fas L suggest that, as a stress inducible protein, HO-1 might be part of the protective mechanisms elicited by the acute phase response in Schistosoma mansoni infection possibly by protecting the cells against the consequences of oxidative stress (i.e. apoptotic and necrotic pathways). HO-1 gene expression may play a role in the modulation of the immunopathology of schistosomiasis and hence, indirectly influence fibrogenesis and prevent cirrhosis.

Inhibitory Effects of Cytotoxicity of Cytokine Activated and Expanded Human CD8+ T Cells by a Human NKG2D Alternative Splice Variant.

NKG2D is an NK cell activating receptor that is also expressed on CD8+ T cells and γδ T cells where it up-regulates activation through TCR co-stimulation. NKG2D is triggered by ligands that are structurally related to MHC1 and expressed either by normal tissues early in ontogeny or by stress, infection or malignant transformation. The function of NKG2D on NK cells and CD8+ T cells is mediated by two distinct pathways of signaling through its association with two adapter proteins, DAP10 and DAP12. Two NKG2D isoforms that preferentially bind to either DAP 10 or DAP12 have been described in murine species. In evaluating human NKG2D gene expression by RT-PCR in cytokine activated CD8+T cells derived from 7 different donors, we consistently observed two distinct PCR bands differing by 218bp. Cloning and sequencing of both PCR amplicons revealed a previously described alternative spliced NKG2D variant resulting from inclusion of intron 6. The intron 6 inclusion introduces a stop codon almost immediately after the start of the intron 6 sequences resulting in a predicted protein product truncated without an extracellular domain. To determine the functional consequences of truncated NKG2D expression we isolated and cloned both full length NKG2D and truncated NKG2D into pRJ3 retroviral vector for expression as NKG2D full length with dsRed and NKG2D truncated EGFP fusion proteins. Activated human CD8+ T cells were trandused and FACS-sorted for dsRed or GFP+ cells and used against human cell line targets in Cr51 release cytotoxicity assays. We found that expression of the full length NKG2D dsRed resulted in ~15% increase in cytotoxicity while expression of the truncated NKG2D GFP resulted in ~70% (p.0009) reduction in cytotoxicity. Results were controlled for cytotoxicity obtained against the same tumor targets using non treated CD8+ T-cells as well as CD8+ T-cells transduced with an empty vector.To further elucidate the role of each NKG2D isoform, we co-expressed both full length NKG2D and truncated NKG2D-EGFP with either DAP-10 or DAP-12. A similar cloning strategy was employed to generate a retroviral expression vector for DAP10-dsRed, DAP10-gfp, DAP12-dsRed and DAP12-gfp fusion proteins. Co expression of NKG2D with dsRed with either DAP10-gfp or DAP12-gfp resulted in comparable 20–25% increase in cytotoxicity (p=.0001) verses NKG2D alone. Co expression of the full length and truncated form also resulted in significant reduction in cytotoxicity of up to 65% (p.0001). Coexpression of the truncated NKG2D-gfp isoform with DAP10-dsRed or DAP12 dsRed did not alter the reduction in cytotoxicity observed with truncated NKG2D alone. We also performed similar experiments using murine CD8+ T cells with human cell line targets RPMI and U266. erexpression of full length NKG2D resulted in >32% increase in cytotoxicity while co-expression of full length NKG2D and DAP10 resulted in 42% increase in cytotoxicity against these human target cell lines. Coexpressing truncated NKG2D and DAP12 resulted in increased cytotoxicity slightly above the background and always less than full length NKG2D. Addition of DAP 10 and DAP 12 to the truncated NKG2D had a minimal impact on cytotoxicity. These results show that the NKG2D isform lacking the extracellular domain inhibits cytotoxicity of activated human CD8+ T-cells. Studies are ongoing to distinguish whether cytotoxocity inhibition is caused by hetrodimerization of the full length and truncated NKG2D incapable of ligand binding, or constitutive and competitive binding by truncated NKG2D of adaptor signaling.

Intraclonal Immunoglobulin VDJ Diversification in Chronic Lymphocytic Leukemia Cells.

The monoclonal Ig expressed on CLL cells was considered to be identical in all cells of the leukemic clone in a CLL patient. However, intraclonal VDJ gene diversification was recently reported in CLL B cells using SSCP analysis and sequencing of Ig PCR products exhibiting altered mobility. We used a different approach, by cloning directly monoclonal VHDHJH PCR bands from CLL patients and sequencing a large number of clones. Peripheral blood from five patients with typical CLL was used to extract DNA.. In one patient DNA from bone marrow and a lymph node biopsy was also analysed. PCR was performed with VH family specific primers and a consensus JH primer for 30 cycles. Monoclonal bands were ligated to TOPO vector and 25 to 70 insert containing clones were sequenced from both orientations. Sequenses contigs were aligned with DNASTAR and were compared with the germ line counterpart using BLAST and IMGT data bases. Intraclonal base variation was observed in 8–33% of the clones in all five patients. One to five base point-mutations /clone were found and there were mostly transitions (in 79% of mutations). In one patient with two monoclonal Ig sequences, (one hypermutated V3-7 and one unmutated VH3-23) intraclonal variation was observed in 69% and 8% of the clones respectivelly. In two patients clones with partially shared mutations were observed indicating the further diversification of a parental subclone. In the patient that VHDHJH clones from blood/bone marrow/lymph node were obtained, a difference in the frequency of diversified clones was noted. All samples were obtained from the patient within one month to exclude the possibility of time related mutations in the monoclonal Ig of the patient. In the patient's VH1-69 unmutated sequence, 19 of 64 (29,6%) of bone marrow derived clones had additional mutations but they were found in only 12 of 71 (16%) peripheral blood derived clones. Five of 53 (9,4%) lymph node derived clones had additional point mutations. Low R/S ratio and targeting of FR rather than CDR regions is against the hypothesis of antigen driven somatic mutations in a fraction of the CLL cells in a given patient. The difference in diversification rate between different tissues in the same patient raises the question of the role of the patient microenvironment/stroma in partcipating in these mutations observed in CLL subclones. With the analysis of large number of clones it is possible that all CLL patients may have at least some degree of intaraclonal VHDHJH gene diversification.

Ecological and biogeographic relationships of class Flavobacteria in the Southern Ocean

The abundance, spatial distribution and diversity of class Flavobacteria were investigated in the Southern Ocean euphotic zone across a latitudinal transect and in the ice pack off Eastern Antarctica. Surface seawater samples filter-fractionated into 0.8 μm particulate and 0.2 m planktonic fractions were investigated with different molecular techniques. The abundance of particle-associated Flavobacteria, ascertained with real-time PCR and DGGE band analysis using Flavobacteria-specific primers, was found to be significantly higher in Polar Front Zone (PFZ) and Antarctic Zone (AZ) water samples than in nutrient limited Temperate Zone (TZ) and Sub-Antarctic Zone (SAZ) waters. Abundance of particle-associated Flavobacteria correlated positively with seawater chlorophyll a and nutrient concentrations, suggesting that increased Flavobacteria abundance may relate to enhanced primary production in the PFZ and AZ. This is supported by comparison of DGGE profiles that demonstrated significant differences in the total Flavobacteria community structure and 16S rRNA gene diversity between samples from the PFZ and AZ and those from TZ and SAZ. Sequence analysis revealed a broad diversity amongst class Flavobacteria in the Southern Ocean with several Flavobacteria clades detected in PFZ and AZ waters not detected in TZ and SAZ waters that putatively represent psychrophilic taxa. Sequence data included a large, so far uncultivated, cosmopolitan phylogenetic clade (“DE cluster 2”) that is distributed throughout the Southern Ocean.

RDNA-ITS 분석에 의한 망태버섯속균(Dictyophora spp.)의 종간 구분 가능성

본 연구는 망태버섯속(Dictyophora species) 균의 종 구분을 위하여 국내수집 5균주와 국외도입 6균주에 대한 rDNA ITS 영역의 PCR-RFLP 및 염기서열을 분석하였다. 그 결과, ASI 32001, 32003, 32005, 32006, 32011이 D. indusiata, ASI 32002, 32007, 32008, 32010이 D. echinovolvata, 그리고 ASI 32004와 Phallus rugulosus ASI 25007은 같은 군으로 그룹화 되었다. 이 결과는 기존에 조사된 배양온도, 배지pH, 선택배지 등 배양적 특성과 재배적, 형태적 특성에 의한 구분과 일치하는 경향이었다. 따라서 rDNA ITS 영역의 PCR-RFLP 및 염기서열분석은 Dictyophora속 보존균주의 종 구분에 있어서 선행적, 보충적 수단으로 활용하기에 충분하다고 판단된다. To establish the phylogenetic relationships of Dictyophora spp., rDNA-ITS regions of 11 strains of veiled lady mushroom collected from various countries were amplified and sequenced. It was observed that the 11 strains were divided into four groups based on PCR band patterns of each ITS region cleaved by eight different restriction enzymes in cleaved amplified polymorphic sequence analysis (CAPS). The phylogenic relationship of each group by cleaved amplified polymorphic sequence (CAPS) analysis matches well with previously reported morphological phylogeny, such as 5 strains of D. indusiata, 4 strains of D. echinovolvata, and a strain of Phallus rugulosus. Sequence analysis using the cluster V methods showed more detail classification than CAPS analysis. The 5.8S region showed two point nucleotide base exchanges from G to A according to four groups, and four groups were subdivided by sequence variation of ITS I and ITS II regions. But sequence variation of Phallus rugulosus was not showed in full ITS region. This study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequence, are most useful in systematics and other mushroom study.

Novel germ-line deletion of SNF5/INI1/SMARCB1 gene in neonate presenting with congenital malignant rhabdoid tumor of kidney and brain primitive neuroectodermal tumor.

We describe a neonate who had a rare tumor combination of a malignant rhabdoid tumor of the kidney (MRTK) and a brain primitive neuroectodermal tumor (PNET). Genetic alterations of the SNF5/INI1/SMARCB1 gene were investigated by PCR-single-strand conformation polymorphism (SSCP), loss of heterozygosity (LOH), sequence, and karyotyping analyses, and the gene expression level was determined by real-time quantitative RT-PCR analysis. PCR band signals of each exon of the hSNF5/INI1 were weak or nearly undetectable in both MRTK and PNET, whereas those of the corresponding normal kidney were clearly detected. Aberrantly migrating SSCP bands led to identification of a nucleotide change in intron 8. Although this was regarded as a polymorphism, only the changed nucleotide was observed in the normal kidney of the patient. Allelic states in the parents were heterozygous for the polymorphism in the father and homozygous for the normal sequence in the mother. Thus, it was evident that a substantial genetic part of the maternal normal allele including SNF5/INI1 was deleted as a de novo germ-line mutation. In both tumors, LOH at microsatellite loci on the long arm of chromosome 22 was evident, and expression of SNF5/INI1 mRNA was drastically decreased compared to that in control tissues (0.7-3.9 vs. 123.6-153.5). Deletion of a substantial genetic part demonstrated in our patient is the novel appearance of a germ-line deletion of the SNF5/INI1 gene. Additional large somatic deletions resulted in total inactivation of the gene in both tumors. Our patient provides evidence for an important role of SNF5/INI1germ-line mutation in predisposing patients to multiple rhabdoid tumors.

Crystalline nickel sulfide-induced genomic instability in transformed human broncho-epithelial cells

To detect the genomic instability in the 16 human broncho-epithelial (16HBE) cells induced by crystalline nickel sulfide so as to provide the scientific basis for further study of nickel-induced cancer molecular mechanism. To analyse the genomic instability in transformed 16HBE cells induced by crystalline nickel sulfide by random amplified polymorphic DNA (RAPD). All the 7 random primers selected could amplify 1 - 6 clear PCR bands. There were no significant differences between transformed 16HBE cells and negative control cells in the 4th, 5th, and 7th primers, but in the rest 4 primers there were significant differences, with special PCR bands for the same primer, indicating that genomic instability in transformed 16 HBE cells was induced by crystalline nickel sulfide. Crystalline nickel sulfide could induce genomic instability in 16HBE cells.

Species-specific polymerase chain reaction primer sets for the diagnosis of Tenacibaculum maritimum infection

In this study the specificity and sensitivity of 2 primer pairs, MAR1-MAR2 and Mar1-Mar2, for the detection of Tenacibaculum maritimum were evaluated in parallel using 79 T. maritimum strains isolated from different fish species, as well as 53 representatives of related and unrelated bacterial species. Both primer pairs were species-specific for T. maritimum, since no amplification products were obtained from chromosomal DNA of the non-T. maritimum bacteria tested. However, whereas MAR1-MAR2 identified all the T. maritimum strains studied, producing a unique and clear PCR band of the expected 1088 bp length, the Marl-Mar2 primer pair failed to amplify the 400 bp specific band in 3 sole isolates. To verify if these strains belonged to T. maritimum species, 2 endonucleases (PvuI and SacII) were selected as the most adequate enzymes to confirm the specificity of the MAR1-MAR2 amplified fragment. The digestion patterns obtained with both endonucleases supported the assignation of all the strains to T. maritimum. The sensitivity of both PCR detection methods was also different, showing a reduction of sensitivity in at least one order of magnitude of the Marl-Mar2 primer pair in comparison with MAR1-MAR2. When the MAR-MAR2 PCR protocol was applied to different seeded turbot tissues, the detection limit was 10(2) to 10(4) T. maritimum cells per reaction. In addition, a nested PCR protocol for detection of this pathogens based on MAR1-MAR2 was developed, which increased the sensitivity by approximately 2 orders of magnitude, ranging from 1 to 250 T. maritimum cells per reaction depending on the tissue employed. The tissues that allowed the most easy detection of T. maritimum were the skin and mucus. Based on the findings reported here, we propose the nested PCR protocol as the most adequate for an accurate detection of T. maritimum in diagnostic pathology as well as in epidemiological studies of gliding bacterial disease of marine fish.

Phylogenetic Analysis and Rapid Detection of Genus Phellinus using the Nucleotide Sequences of 18S Ribosomal RNA

Analysis of phylogenetic relationship was performed among Phellinus species based on 18S ribosomal subunit sequence data. Twenty-five strains of 19 Phellinus species including P. linteus were examined in this study. Regions of 18S ribosomal subunit were very conserved, but some variable regions between Phellinus species were observed. The species-specific detection primers, modified by 2 or 3 nucleotides in sense primer were designed based on 18S ribosomal DNA (rDNA) sequence data. The 210 bp PCR bands were detected with annealing temperature 48°C. The 18S 2 F-18S 4 R detection primer set distinguished P linteus from various Phellinus species but some species like P baumii, P weirianius, P rhabarberinus and P pomaceus also had weak reactivity on this primer set. The 18S 3 F-18S 4 R primer set distinguished only P. linteus from various Phellinus species, although sensitivity with this primer set was lower than that of 18S 2 F-18 4 R primer set. These primer sets would be useful for the detection of only P. linteus among unknown Phellinus species rapidly.

Physical Mapping of the Bovine Immunoglobulin Heavy Chain Constant Region Gene Locus

Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.

Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit.

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3′ end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from “stem cell genes” being highly expressed at 0 h and turned off at 48 h, while “cell division” genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

Thyroid transcription factor-1 expression in the human neonatal tracheoesophageal fistula

Background/Purpose: Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a relatively common congenital anomaly, but its pathogenesis remains poorly understood. Previous studies using the experimental Adriamycin-induced rat model of EA/TEF suggest that the fistula tract, or [ldquo ]distal esophagus,[rdquo ] is derived from respiratory epithelium and expresses the respiratory-specific transcription factor TTF-1. To better correlate the experimental rat model of EA/TEF with the human anomaly, we looked for evidence of a respiratory-derived origin in the neonatal TEF. Methods: After IRB approval, 2 human TEF samples were removed at the time of surgery. The tissue from the fistula tract was trimmed in accordance with what the surgeons deemed to be appropriate for the preparation for a primary anastomosis. The tissues then were processed for reverse transcription polymerase chain reaction (RT-PCR), histology, and immunohistochemistry. Normal embryonic lung cDNA was used for positive controls. Results: Histologic examination of tissue specimens showed many epithelial tubules within loose connective tissue and a disorganized muscular coat. Thyroid transcription factor one (TTF-1) and hepatocyte nuclear factor 3[beta ] (HNF-3[beta ]) were shown to be present in the tissue specimen by RT-PCR and immunohistochemistry. In addition, FGF-10, a strong morphogen present in the developing lung, and FGF-1 were both present by RT-PCR. The PCR band sizes for both FGF-1 and -10 were appropriate, using human embryonic cDNA as a control, and the bands were confirmed as nongenomic by either placing the PCR primers across a known intron sequence (TTF-1) or by absence of a band in a negative RT control (HNF-3[beta ], FGF-10, FGF-1). Conclusions: The presence of TTF-1 in the neonatal TEF shows, for the first time, that parallels may be drawn between the experimental rat model of TEF and the human anomaly at the molecular level. Moreover, these results suggest that the fistula tract in the human neonate is derived from a respiratory cell lineage. This respiratory origin of the human TEF may explain the poor esophageal motility (and subsequent serious respiratory complications) of the distal segment after standard EA/TEF repair. J Pediatr Surg 37:1065-1067.

Analysis of T cell receptors reactive with squamous cell carcinoma antigen SART-1 presented by the HLA-A24 molecule

We investigated the induction of cytotoxic T lymphocytes (CTLs) specific for squamous cell carcinoma antigen SART-1 from the peripheral blood mononuclear cells (PBMCs) of an HLA-A24 healthy donor by in vitro stimulation with dendritic cells (DCs) pulsed with the antigenic peptide. Effector cells, designated as <peptide-pulsed dendritic cell-activated killer (PDAK)> cells, exhibited potent cytotoxic activity against target cells in a SART-1-specific and HLA-restricted manner. T cell receptor (TCR) gene usage of the SART-1 PDAK cells analyzed by reverse transcribed-polymerase chain reaction (RT-PCR) and Southern blot analysis showed oligoclonal usage, including TCRVbeta18, which was indicated by blocking assay to be responsible for antigen recognition and killing. Single strand conformation polymorphism (SSCP) analysis revealed clonotypic bands of the TCRVbeta18. The PCR product of the TCRVbeta18 was sequenced and complementarity determining region (CDR) 3 was determined. RT-PCR analysis using this CDR3 sequence as a primer showed, as expected, a positive band of about 30 bp smaller than shown on regular PCR analysis. This clonotypic PCR demonstrated an increasing density of PCR bands in the SART-1 PDAK cells during stimulation with SART-1 peptide plus DCs. It is suggested that clonotypic PCR using the TCR-CDR3 sequence may be useful in assessing the precursor frequency of CTLs reactive with the SART-1 peptide in vivo.

PCR identification of dermatophyte fungi Trichophyton rubrum, T. soudanense and T. gourvilii.

Diagnosis of dermatophytosis employing conventional laboratory procedures has been complicated by the slow growth and varied morphological features shown by dermatophytes. After analysis of the nucleotide base sequences of a 1.2-kb fragment amplified from a dermatophyte fungus Trichophyton rubrum by arbitrarily primed PCR with random primer OPD18, a pair of primers (TRIF and TR1R) was designed and evaluated for specific identification of T. rubrum. The sensitivity of the primers TR1F and TR1R was high, as a specific PCR band of c. 600 bp was detected from as little as 7 pg of T. rubrum DNA. By examining 92 dermatophyte strains and clinical isolates, it was found that this pair of primers reacted in PCR with T. rubrum, T. soudanense and T. gourvilii through formation of the specific fragment of 600 bp, but not with any other of the dermatophyte species or varieties, fungi, yeasts or bacteria tested. As T rubrum is one of the most frequently isolated dermatophyte fungi, and T. soudanense and T. gourvilii are relatively uncommon in many parts of the world, these primers can be used for rapid, sensitive and specific identification and differentiation of T. rubrum from other fungi and micro-organisms.

First Report of a Distinct Begomovirus Infecting Cassava from Zanzibar.

In 1998, cassava plants exhibiting extremely mild mosaic disease symptoms were collected from Uguja Island, Zanzibar. Total DNAs extracted from symptomatic leaves did not produce diagnostic PCR bands using primers specific to known Cassava mosaic viruses (genus Begomovirus, family Geminiviridae) (2). Degenerate primer pair A/B (1), however, produced a 564-nucleotide (nt) band in the common region of DNA-A to the conserved amino acid sequence CEGPCKYG within the coat protein gene for begomoviruses. Virus-specific primers were designed and the begomoviral genome was amplified and cloned to obtain the full-length DNA-A (2,785 nt) (AF422174) and the partial DNA-B (2,403 nt) (AF422175) component comprising the 5' end of the BV1 open reading frame (ORF) to eight (A) of the conserved nanonucleotide TAATATTAC in the common region. Phylogenetic comparisons of DNA-A and -B sequences of the cassava begomivirus showed they were most closely related to East African cassava mosaic virus (EACMV) from Tanzania (Z83256, 83% identity) and Uganda (AF126805, 87% identity). Based on DNA-A and -B components, its closest relatives among African cassava mosaic virus strains were the Nigerian (X17095, 67% identity) and Ugandan (AF126800, 38% identity) isolates respectively. The number and arrangement of the viral ORF was identical to other bipartite begomoviruses from the Old World. Work is in progress to determine whether this begomovirus is a new strain of an existing EACMV or a new species.