Polymerase Chain Reaction Amplification Of DNA Research Articles

Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica

The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z 4,z 23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382–384 bases in length, which were 98.4–99.7% similar to each other and three long fragments of 505 bases with 98.0–99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

Analysis of p53 and mdm2 proteins in malignant fibrous histiocytoma in absence of gene alteration: prognostic significance.

TP53 and MDM2 genes and their protein expression were evaluated in frozen and paraffin-embedded tissue from 27 patients with malignant fibrous histiocytoma to elucidate the relationship between them, their implication in tumor progression mechanisms and their possible diagnostic-prognostic value in malignant fibrous histiocytoma. Single-strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction-amplified DNA were used to establish two TP53 mutations (7.4%): a point mutation and a 63-bp duplication. Amplification of the MDM2 gene was observed in two tumors (7.4%) by means of Southern-blot analysis, one of them also carrying the TP53 point mutation. Immunohistochemical and Western-blot techniques were used to study nuclear accumulation of p53 and mdm2 proteins: 11 cases (40.7%) with p53 protein expression and thirteen cases (48.1%) with mdm2 protein expression were detected. We confirmed overexpression of mdm2 protein in eight of ten cases (80%) with p53 protein expression without TP53 gene mutation. Statistical analysis shows that simultaneous co-expression of p53 and mdm2 in malignant fibrous histiocytoma is significantly correlated with survival in absence of gene alteration in contrast to the lack of statistical correlation with survival of p53 protein expression alone.

A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue

We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.

Adeno-associated virus and development of cervical neoplasia.

Evidence from several sources has suggested that adeno-associated virus (AAV) infection might protect against cervical cancer, in part, by interfering with human papillomavirus (HPV)-induced tumorigenesis. Detection of AAV type 2 (AAV-2) DNA in cervical tissues has been reported. However, there have been few in vivo studies of women with cervical HPV infection or neoplasia, and these have reported inconsistent results. Therefore, we used polymerase chain reaction (PCR) assays targeted to the AAV-2 rep and cap genes to test tissue specimens from women in an epidemiological study of cervical neoplasia in Jamaica. We tested 105 women with low-grade cervical intraepithelial neoplasia (CIN-1), 92 women with CIN-3/carcinoma in situ or invasive cancer (CIN-3/CA), and 94 normal subjects. PCR amplification of human beta-globin DNA was found in almost all cervical specimens, indicating that these materials were adequate for PCR testing. The prevalence of HPV DNA, determined by HPV L1 consensus primer PCR was, as expected, strongly associated with presence and grade of neoplasia. Each of the AAV PCR assays detected as few as 10 copies of the virus genome. However, none of the 291 cervical specimens from Jamaican subjects tested positive for AAV DNA. Negative AAV PCR results were also obtained in tests of cervical samples from 79 university students in the United States. Exposure to AAV was assessed further by serology. Using a whole virus AAV-2 sandwich enzyme-linked immunosorbent assay, we found no relationship between AAV antibodies and presence or grade of neoplasia in either the Jamaican study subjects or women enrolled in a U.S. cervical cancer case (n = 74) -control (n = 77) study. Overall, the data provide no evidence that AAV infection plays a role in cervical tumorigenesis or that AAV commonly infects cervical epithelial cells.

Deletion mapping at 12p12-13 in metastatic prostate cancer

The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.

Differences among Helicobacter pylori strains isolated from three different populations and demonstrated by restriction enzyme analysis of an internal fragment of the conserved gene hpaA.

Our goal was to test the idea that Helicobacter pylori genotypes vary from one population to another. Analysis of Sau3A and HinfI restriction fragment-length polymorphism (RFLP) in a 375-bp polymerase chain reaction amplicon of hpaA was used to compare 31 H. pylori isolates from a relatively small and genetically homogeneous population (Goteborg, Sweden) with those of large, genetically heterogeneous populations located in two different countries (50 isolates from Houston, TX, and 69 isolates from Minas Gerais, a state in the southeastern region of Brazil). Five different Sau3A and three different HinfI restriction patterns were found; different combinations of these comprise 10 different RFLP types, I through X. The RFLP types found in the United States and Brazil collections were very similar, except for two Brazil isolates belonging to type VIII and five Brazil isolates belonging to type X, neither type found in the United States. The overall profile of H. pylori isolates from Sweden was remarkably different, with 18 of 31 (58%) having a new Sau3A restriction pattern, termed gS; 10 of these 18 isolates had HinfI restriction pattern E (RFLP type VIII), and 8 had HinfI restriction pattern F (RFLP type IX). No isolates from Sweden belonged to RFLP type III or type X. RFLP typing of a 375-bp polymerase chain reaction-amplified DNA fragment of H. pylori hpaA revealed that H. pylori genotypes can and do vary from one population to another. We conclude that the unique RFLP profile shown by the group of H. pylori isolates from Goteborg is the result of a cohort effect in this relatively small, stable, genetically homogeneous population. Also, the overall similarity between RFLP profiles of the H. pylori isolates from Texas and Minas Gerais coincides with the fact that although geographically distanced, these populations are similar in being large, dynamic, and genetically heterogeneous.

Molecular Phylogenetic Analyses of Biological Control Strains of Trichoderma harzianum and Other Biotypes of Trichoderma spp. Associated with Mushroom Green Mold

ABSTRACT A polymerase chain reaction-amplified DNA containing the internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 regions of the nuclear ribosomal DNA transcriptional unit was sequenced for 81 isolates of Trichoderma spp. associated with mushroom culture or used for biological control of plant pathogens. Phylogenetic analyses revealed that the biocontrol isolates were more closely related to an isolate of T. harzianum biotype 1 (Th1) than to the aggressive biotypes 2 and 4. Th1 has been isolated from mushroom compost but is not the cause of widespread green mold epidemics that have occurred during the last 12 years in Europe and North America. Three isolates of T. harzianum obtained from shiitake (Lentinula edodes; Shi1B and S3-96) and maitake (Grifola frondosa; Mai1) substrates were placed within the biocontrol group. We also found evidence suggesting that some isolates of T. harzianum originally identified as Th4 from Pennsylvania are more closely related to Th2 from Europe. Finally, considering the wide range in sequence distribution of our samples, we propose that the consensus sequence found in this investigation be used as the reference sequence for further studies involving the identification and taxonomy of T. harzianum.

Perinatal cytomegalovirus infection: commonly occurring but rarely diagnosed

OBJECTIVE: To determine the incidence rate of perinatal cytomegalovirus (CMV) infection and to describe the clinical presentation of this infection in non-transfused term infants attended at a general hospital, in Ribeirão Preto, SP, Brazil. POPULATION AND METHODS: Thirty four infants free of CMV infection at birth were followed up during the first 4 months of life. Diagnosis of perinatal CMV infection was established by isolating the virus in tissue culture or by polymerase chain reaction DNA amplification (PCR) in urine samples. RESULTS: A 38.2% (13/34) incidence rate of perinatal CMV infection was detected, and only 3 of the infected infants were symptomatic (respiratory tract symptoms in one infant and splenomegaly in two). CONCLUSIONS: The results indicate a high incidence rate of perinatal CMV infection in the studied infants. Clinical symptoms were present in 23% of these patients, stimulating the investigation of this agent as a cause of pneumonitis and splenomegaly.

Genetic variation between strains of Monilinia fructicola and Monilinia laxa isolated from cherries in Michigan

Polymerase chain reaction (PCR)-mediated analysis of rDNA from isolates of Monilinia fructicola and Monilinia laxa from Michigan cheny orchards revealed interspecies restriction site variation in the internal transcribed spacer 1 (ITSI) region and length variation in the small subunit (SSU) rRNA gene. 1TS1 sequences from both species were 146 by long; however, the ITSI of M. laxa differed at three positions from the ITSI of M. fructicola. Although the sequences of the ITS1 regions from both species were nearly identical, the enzyme tilrel cuts the PCR-amplified ITSI region of the two species differentially. PCR amplification of the 3′ end of the SSU rRNA gene yielded products of approximately 940 and 520 by from M. fructicola and M. laxa, respectively. A 421-bp group I intron was detected by PCR within the SSU rDNA of 32 isolates of M. fructicola but not in the eight isolates of M. laxa. Intron sequences from each of four isolates of M. fructicola were identical, and the SSU rDNA flanking sequences from these isolates and from two isolates of M. laxa were nearly identical. Arbitrarily primed PCR analysis of genomic DNA with microsatellite primers (GACA)4 and (GTG)5 revealed that the number and size of the amplification products were chazacteristic for each species. Distinctive and reproducible sets of amplification products were obtained from 32 isolates of M. fructicola and the eight isolates of M. laxa. Our results illustrate the potential of PCR amplification of ribosomal and genomic DNA for differentiating these tree-fruit-infecting brown rot fungi.

Prevalence and clinical aspects of congenital cytomegalovirus infection

OBJECTIVES: The objectives of the present study were to determine the prevalence of congenital CMV infection, as well as to evaluate the importance of this agent as cause of congenital disease, and to describe the clinical manifestations in children attended at a General Hospital in Ribeirão Preto, SP, Brazil. POPULATION AND METHODS: A first group including 189 newborns and their mothers was evaluated for the prevalence of the congenital CMV infection. A second group including 130 newborns and 74 infants who presented clinical manifestations of congenital disease were also investigated to evaluate the importance of the CMV as a cause of this disease and to describe the clinical findings. Diagnosis of congenital CMV infection was established by detecting the virus using viral isolation in tissue culture, polymerase chain reaction DNA amplification in urine samples and detection of specific anti-CMV IgM and IgG by immunofluorescence indirect test. RESULTS: The prevalence of congenital CMV infection was 2.6% and the prevalence of CMV antibodies in mothers was 95%. In the first group, none of the 5 congenitally infected presented clinical apparent disease at birth, although one of them had intracranial calcifications. In the second group, CMV was recognized as a causative of congenital disease in 12 children (5.9%). Of these, 10(83%) were identified after the neonatal period. The clinical findings included hepatosplenomegaly (75%), jaundice with direct hyperbilirubinemia (42%), neurologic disease consisting of microcephaly and intracranial calcifications in 42% of these children. CONCLUSIONS: The prevalence of congenital CMV infection was similar to that reported in other studies about highly immune populations. Infants with asymptomatic congenital CMV infection may have diseases of the central nervous system that are not clinically evident at birth, such as punctate calcifications. CMV infected patients who are symptomatic at birth have a multisystem disease, and the differential diagnosis of any newborn with clinical abnormalities including involvement of the hepatobiliary, hematopoietic and central nervous systems should include congenital CMV infection. CMV was an important agent of these abnormalities, and the majority of symptomatic patients were identified after the neonatal period, making the diagnosis more difficult.

Clonal mixing, clonal restriction, and specification of cell types in the developing rat olfactory bulb

To understand the clonal relationship of various olfactory bulb (OB) cell types, OB progenitor cells were infected at embryonic day (E) 14, E15, and E17 with retroviral libraries encoding alkaline phosphatase or beta-galactosidase. After survival to postnatal day 10-15, sibling relationships were identified by polymerase chain reaction DNA amplification of distinct sequences in the retroviral constructs. Within the OB, clonal progeny dispersed widely in all directions. In sharp contrast, however, clonal dispersion between the OB and neocortex was not observed, although occasional clonal dispersion between the OB and pyriform and hippocampal regions could not be excluded. Most clones (84%) contained a single cell type, especially after E17 injections, suggesting the existence of either restricted precursors, or multipotential progenitors instructed by a restricted cellular environment. Mixed OB clones (16%) contained multiple cell types in the OB, or occasionally glial or neuronal cells outside the OB, demonstrating the existence of multipotential OB progenitors, likely at a stage before formation of the olfactory rostral migratory stream. Surprisingly, OB glial cells were not labeled, suggesting distinct lineages or perhaps distinct migratory paths for glia and neurons into the OB. A hierarchical cell lineage is proposed that involves a multipotential progenitor that gives rise to potentially more limited progenitors.

Analysis of DNA from A Single Barley Embryo by the DNA Polymerase Chain Reaction (PCR) Technique

This study was conducted to investigate whether DNA extracted from a single barley embryo is suitable for conducting polymerase chain reaction (PCR) amplification. We extracted DNA from one-seed and five-seed samples of four barley cultivars, ‘Bowman’, ‘Colter’, ‘Crystal’, and ‘Russell’. Six random-amplified polymorphic DNA (RAPD) primers and one sequence-tagged-site (STS) primer pair were tested for DNA amplification using both Taq DNA polymerase and the Stoffel fragment, which is a modified form of recombinant Taq DNA polymerase. DNA polymorphism was found with the STS primer and five of the RAPD primers. The banding patterns of DNA from one-seed samples were almost identical to those of the five-seed samples. Additional testing of nine barley cultivars was conducted to compare embryo and leaf tissue DNA PCR amplification results. Our tests indicated that DNA extracted from a single embryo is practical for PCR analysis. The technique we utilized is simple, fast, and can be applied to the identification of barley cultivars.

Cloning and Expression of cDNA for a Human Siaα2,3Galβ1,4GlcNA:α2,8-Sialyltransferase (hST8Sia III)

The cDNA encoding human Sia-α2,3-Gal-β1,4-GlcNAc-R:α2,8-sialyltransferase, hST8Sia III, was isolated by screening of a human brain cDNA library with polymerase chain reaction-amplified DNA probe generated from the sequence of mouse ST8Sia III (mST8Sia III) and by 5′ rapid amplification of cDNA ends of mRNA isolated from human brain tissues. Comparative analysis of the predicted protein-coding region between our cloned hST8Sia III and mST8Sia III showed 92 and 96% identities in the nucleotide and the amino acid sequence, respectively. The soluble hST8Sia III protein expressed in COS-7 showed an extremely high catalytic activity of transferring sialic acid through α2,8-linkage to intact fetuin glycoprotein, whereas the transferring activity was completely undetectable toward either α2,6-sialylated glycoprotein or desialylated glycoprotein acceptors. Northern analysis of hST8Sia III showed that the transcript corresponding to 11 kb was expressed in both human fetal and adult brain, while the expression of the 5.5-kb transcript was restricted to fetal liver, indicating that the expression of hST8Sia III is developmentally and tissue-specifically regulated.

PCR amplification of DNA obtained from archived hematoxylin-eosin--and giemsa-stained breast cancer aspirates.

The study of DNA abnormalities in fine-needle aspiration (FNA) specimens for breast cancer could be helpful in improving the capacity of diagnosis and specially to obtain prognostic or predictive information. The aim of the present study was to verify whether it is possible to perform molecular analysis in slides of breast cancer FNA specimens. previously stained by hematoxylineosin (H&E) and Giemsa, stored at least for 3 years. For this purpose, 10 cases of FNA obtained from breast cancer patients diagnosed between 1993 and 1994 in our institute were used. Five cytologic smears were alcohol-fixed and stained with H&E. The other five were air-fixed and Giemsa stained. DNA was isolated from the cytologic smears and amplified by using radioactive polymerase chain reaction (PCR) aimed at BAT-26 marker. Our results demonstrate that archived stained smears prepared for cytologic examinations can be used for molecular analyses by using a PCR amplification method. DNA could be isolated and PCR amplified independently of the prior fixation and staining procedures. So, we conclude that the application of molecular biology techniques to the existing archival smears may become a valuable tool to detect genetic changes in samples from breast cancer aspirates.

PCR amplification of DNA obtained from archived hematoxylin‐eosin– and giemsa‐stained breast cancer aspirates

The study of DNA abnormalities in fine-needle aspiration (FNA) specimens for breast cancer could be helpful in improving the capacity of diagnosis and specially to obtain prognostic or predictive information. The aim of the present study was to verify whether it is possible to perform molecular analysis in slides of breast cancer FNA specimens, previously stained by hematoxylin-eosin (H&E) and Giemsa, stored at least for 3 years. For this purpose, 10 cases of FNA obtained from breast cancer patients diagnosed between 1993 and 1994 in our institute were used. Five cytologic smears were alcohol-fixed and stained with H&E. The other five were air-fixed and Giemsa stained. DNA was isolated from the cytologic smears and amplified by using radioactive polymerase chain reaction (PCR) aimed at BAT-26 marker. Our results demonstrate that archived stained smears prepared for cytologic examinations can be used for molecular analyses by using a PCR amplification method. DNA could be isolated and PCR amplified independently of the prior fixation and staining procedures. So, we conclude that the application of molecular biology techniques to the existing archival smears may become a valuable tool to detect genetic changes in samples from breast cancer aspirates. Diagn. Cytopathol. 1998;19:395–397. © 1998 Wiley-Liss, Inc.

Familial migraine with vertigo: no mutations found in CACNA1A.

We searched for mutations in the voltage-gated calcium channel gene, CACNA1A, in nine propositi of families with migraine headaches and episodic vertigo inherited in an autosomal dominant pattern. All 47 exons and flanking introns in CACNA1A were subjected to single-strand conformation polymorphism analysis of polymerase chain reaction-amplified genomic DNA. Exons with aberrantly migrating fragments were sequenced using standard techniques. We also determined the CAG repeat length at the 3' end of CACNA1A. Several polymorphisms were found but no mutations identified in any of the 47 exons of the 9 patients. No index-case had a CAG repeat length greater than 13 (normal <17). Mutations in CACNA1A are not common in families with migraine headaches and episodic vertigo. Other ion channel genes expressed in the brain and inner ear remain candidate genes.

A taxon-specific oligonucleotide probe for temperate zone soil isolates of Glomus mosseae

The 5.8 S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus mosseae FL156 and UK118 were amplified by polymerase chain reaction (PCR) using ITS1 and ITS4 as primers. The amplification product from template DNA of UK118 was cloned and sequenced (569 bp); the amplified DNA from FL156 was sequenced directly (582 bp). There was a 95% sequence similarity between DNAs amplified from the two isolates; in contrast, major dissimilarities with partial sequences of seven other glomalean taxa were observed. Four oligonucleotide sequences unique to Glomus mosseae were identified as potential primers. Their specificity to Glomus mosseae was assessed by PCR amplification of genomic DNA from spores from 36 glomalean fungi: 13 isolates of Glomus mosseae, two Glomus monosporum, 10 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The Glomus mosseae isolates were from a broad range of temperate zone agricultural soils. Oligonucleotide pair GMOS1 : GMOS2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all Glomus mosseae isolates and two isolates of the closely related Glomus monosporum. This primer pair did not prime PCR when the template consisted of DNA from any of the other glomalean fungi or any of the nonmycorrhizal controls. In addition, a 24-mer oligonucleotide, designated GMOS5, hybridized with Glomus mosseae and Glomus monosporum DNA amplified by PCR using primer pairs ITS1 : ITS4 and GMOS1 : GMOS2. Colony-blot assays showed that GMOS5 hybridized to 100% and 97% of E. coli pUC19 clones of amplification products from Glomus mosseae FL156 and UK118 DNA templates, respectively, indicating that nearly all clones contained an homologous sequence. GMOS5 was used successfully to detect specifically Glomus mosseae in DNA extracted from colonized sudan grass (Sorghum sudanense L.) roots and amplified by PCR using the primer pair GMOS1 : GMOS2. The results confirm several previous indications that Glomus mosseae and Glomus monosporum are indistinguishable taxonomic entities.

Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. However, this requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid, inexpensive and suitable for diagnosing trisomies, even from single fetal cells, is the fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STRs). In this paper, we present the method of rapid prenatal detection of trisomy 13 from amniotic fluid using fluorescent PCR and two highly polymorphic STRs (D13S258 and D13S631). The results obtained by quantitative fluorescent PCR amplification of fetal DNA were concordant with amniocyte karyotyping results in all cases. Two cases of trisomy 13 were detected from 212 amniotic fluids and the results obtained from D13S631 and D13S258 amplification are presented. In the first trisomy 13 case, a triallelic pattern was detected by both markers, and in the second case, D13 markers showed a characteristic 2:1 dosage allele ratio, both of which demonstrate trisomy 13 status. All other heterozygous disomic samples showed an allele intensity ratio of 1:1.

Isolation of a human thiopurine S-methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity. Radiochemical assay, Western blot analysis, polymerase chain reaction (PCR) genotyping, and cDNA sequencing were used to analyze TPMT activity and protein levels in erythrocytes and to determine TPMT genotype. We established expression of another common mutant allele, TPMT*3C (containing only the A719G mutation), by sequence analysis of cDNA isolated from an individual with a heterozygous TPMT phenotype (7 units/ml packed erythrocytes). The TPMT*3C allele was also confirmed in an unrelated individual by sequencing TPMT coding exons after PCR amplification of genomic DNA. Moreover, Western blot analysis of erythrocytes obtained from five heterozygous individuals with the TPMT*3C allele (i.e., TPMT*1/TPMT*3C) exhibited about 50% less immunodetectable TPMT protein compared with homozygous wild-type individuals, and a TPMT-deficient individual with a TPMT*3A/TPMT*3C genotype had no immunodetectable TPMT protein. These data establish that the TPMT*3C allele is expressed in humans and is associated with lower immunodetectable TPMT protein and catalytic activity.

SPECIFIC PRIMERS FOR RAPID TYPING OF TUBER BORCHII MYCORRHIZAL ROOTS

Truffles are Ascomycetous fungi that need to form a symbiotic association (mycorrhiza) with the roots of a host plant to be able to produce their fruitbodies. Many of the morphological characters used to identify a truffle fruitbody, however, are lost during this phase. DNA analyses have therefore been devised for the typing of truffle species. The paper describes an application of this technique to identify the mycorrhizas of Tuber borchii (a commercial species) by means of the polymerase chain reaction (PCR), this being the most effective way of typing from mycorrhizas or mycelium. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was employed as the target, since it displays a high degree of variability and is highly repeated. The ITS region of T. borchii was cloned and sequenced. A primer pair (TBA/TBB) designed by comparing three Tuber species sequences and employed for PCR amplification of DNA isolated from T borchii fruitbodies, mycelia and mycorrhizas proved to be specific for this species at all three levels of its life cycle. It also failed to amplify DNA from T. maculatum and thus ensured the unambiguous differentiation of a species whose morphology is very similar to that of T borchii.