Sequence Analysis Of Polymerase Chain Reaction Products Research Articles

Deletion mapping of chromosome 16q24 in hepatocellular carcinoma in Taiwan and mutational analysis of the 17-beta-HSD gene localized to the region.

Human chromosome band 16q24 commonly undergoes loss of heterozygosity (LOH) in human hepatocellular carcinoma (HCC). To further localize the region of deletion on 16q24 and to evaluate the genetic role of 17-beta-HSD, which is near 16q24, in HCC, we examined the pattern of loss of heterozygosity in 88 HCC patients. DNAs from 88 pairs of HCCs and corresponding non-tumor parts were prepared. Loss of heterozygosity on chromosomes 16q24 was investigated by 11 sets of microsatellite markers. Mutation analysis of type II 17-beta-HSD was performed by automatic sequencing. LOH on 16q24 for at least 1 locus was found in 43 of the 88 tumor DNAs (49%). Three non-overlapping regions of frequent LOH were defined in these 43 tumors with partial deletions. The first region was between D16S516 loci and D16S507, encompassed by a 1-cM region, defined by the D16S504. The second region was defined by the 17HSDB2 locus between D16S505 and D16S422, encompassed approximately by a 1-cM region. The third region was between D16S520 and D16S413, defined by D16S3048, encompassed approximately by a 4-cM region. Homozygous deletions of any exons in 17HSDB2 gene were identified in 7 of 27 cases (26%). Automated sequencing analysis of 17HSDB2 failed to demonstrate mutations in any of these specimens. Our data suggest that the 17HSDB2 locus is a frequent target of deletion in HCC but the inactivation of 17HSDB2 may not involve sequence mutations. Furthermore, the presence of the other 2 frequent LOH regions suggest that the putative tumor suppressor genes at these locations might be involved in the development of HCC.

Human immunodeficiency viruses type 1 subtypes circulating in Spain

Genetic subtypes of Human immunodeficiency viruses type 1 (HIV-1) were investigated in 101 HIV-1-infected individuals living in Spain from 1993 to 1998. Samples selected randomly from the HIV clinic population included 29 Spanish native born subjects (28.7%) and 72 foreigners (71.3%). Proviral DNA extracted from peripheral blood mononuclear cells (PBMCs) or viral RNA isolated from plasma was amplified, and endonuclease restriction analysis was carried out on polymerase chain reaction (PCR) products. Restriction fragment length polymorphism (RFLP) analysis on the HIV-1 protease region enabled the characterisation of the different HIV genotypes infecting these individuals. Overall, 38 subjects (37.6%) carried non-B subtypes (A in 26, C in 2, D in 1, E in 2, and F in 7), 31 (81. 6%) of them being immigrants. Direct sequence analysis of PCR products and/or a specific serological assay confirmed the data obtained by RFLP in most individuals tested. In conclusion, different HIV-1 subtypes are circulating currently in Spain, with non-B HIV-1 subtypes being confined mostly to immigrants.

Human immunodeficiency viruses type 1 subtypes circulating in Spain

Genetic subtypes of Human immunodeficiency viruses type 1 (HIV-1) were investigated in 101 HIV-1-infected individuals living in Spain from 1993 to 1998. Samples selected randomly from the HIV clinic population included 29 Spanish native born subjects (28.7%) and 72 foreigners (71.3%). Proviral DNA extracted from peripheral blood mononuclear cells (PBMCs) or viral RNA isolated from plasma was amplified, and endonuclease restriction analysis was carried out on polymerase chain reaction (PCR) products. Restriction fragment length polymorphism (RFLP) analysis on the HIV-1 protease region enabled the characterisation of the different HIV genotypes infecting these individuals. Overall, 38 subjects (37.6%) carried non-B subtypes (A in 26, C in 2, D in 1, E in 2, and F in 7), 31 (81.6%) of them being immigrants. Direct sequence analysis of PCR products and/or a specific serological assay confirmed the data obtained by RFLP in most individuals tested. In conclusion, different HIV-1 subtypes are circulating currently in Spain, with non-B HIV-1 subtypes being confined mostly to immigrants. J. Med. Virol. 59:189–193, 1999. © 1999 Wiley-Liss, Inc.

Use of the same archival papanicolanou smears for detection of human papillomavirus by cytology and polymerase chain reaction.

An optimal method for the processing of archival cervical Papanicolaou (pap)-stained smears for the amplification of human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) was developed. This methodology was then applied to a series of 44 pap smears designated as HPV positive or negative (on the basis of both major and minor cytological criteria) or cervical intraepithelial neoplasia (CIN)-cancer. For the detection of HPV DNA, each sample was tested with the consensus GP5/6 primers, and when negative, with CPI-IIG primers. The HPV DNA was detected in 100% (8 of 8) of CIN-cancer smears using the GP5/6 primers. In smears with cytological evidence of HPV without CIN. the use of both sets of primers yielded positive results in 100% (19 of 19) of the samples. Direct sequence analysis of PCR products showed that 16 of the 27 HPV-positive samples contained more recently described HPV types. When tested with both primer combinations, all 17 cytologically negative smears were positive for beta-globin but negative for HPV DNA. The findings show the value of using archival pap smears for further investigations to address issues such as latency, but they indicate that cytological criteria and DNA technology will be critical factors in the reliability of the results.

Atypical immature metaplasia (AIM) of the cervix: Is it related to high-grade squamous intraepithelial lesion (HSIL)?

Atypical immature metaplasia (AIM) is a poorly characterized cervical lesion with uncertain biological and clinical significance. AIM shares some, but not all, morphological features of squamous intraepithelial lesions (SILs). SILs are characterized by human papillomavirus (HPV) positivity and increased cellular proliferation, but these features have not been fully evaluated in AIM. Genomic DNA was extracted from 27 microdissected cervical biopsy specimens diagnosed as AIM. HPV DNA was detected by polymerase chain reaction (PCR), using two different sets of L1 gene consensus primers. HPV types were identified by sequence analysis of PCR products and comparison with published HPV sequences. The cell proliferation index was assessed by immunohistochemical staining for Ki-67 (MIB-1) antigen and expressed as the percentage of Ki-67—positive cells. Comparison groups included normal cervix ( n = 10) and low-grade (LSILs, n = 19) and high-grade squamous intraepithelial lesions (HSILs, n = 11). Intermediate- or high-risk HPV DNA was detected in 67% (18 of 27) of AIM cases. Low-risk HPV DNA was not detected in any of the specimens. The Ki-67 index in AIM (mean, 33.0 ± 20.3; median, 29) was comparable to that of LSILs (mean, 21.4 ± 4.6; median, 21) and was significantly higher than that of normal cervix (mean, 11.0 ± 2.1; median, 11) ( P < .01) and lower than that of HSILs (mean, 60.4 ± 13.2; median, 60) ( P < .01). Of the cases with available follow-up, HPV-positive AIMs were significantly more likely to have a concurrent or subsequent diagnosis of typical HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 45%) ( P = .014). The wide range of Ki-67 indices and variable HPV status in AIM suggest that AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (high-risk HPV-positive, low to moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). HPV testing may be useful in the assessment of atypical epithelial proliferations of the cervix for which a diagnosis of AIM is considered.

A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms

Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n > or = 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.

Protein kinase C and receptor kinase gene expression in olfactory receptor neurons.

Recent biochemical evidence indicates that protein kinase C (PKC) and G-protein-coupled receptor kinases (GRKs) are involved in olfactory signal termination and desensitization. The polymerase chain reaction (PCR) was used to investigate the expression of PKC and GRK genes in olfactory tissue and in isolated olfactory receptor neurons from channel catfish (Ictalurus punctatus). Sequence analysis of cloned PKC PCR products showed that the alpha, beta, delta, epsilon, and theta isotypes were expressed in olfactory tissue. Sequence analysis of PCR products obtained from isolated olfactory receptor neurons showed that PKC beta and PKC delta were expressed in the receptor cells. A 600-bp GRK PCR product was obtained from isolated olfactory neurons that shared 86% and 92% amino acid sequence identity to the mammalian beta-adrenergic receptor kinase gene products beta ARK1 and beta ARK2, respectively. Go6976, a specific inhibitor of calcium-regulated PKC activity, completely inhibited odorant-stimulated PKC activity in isolated olfactory cilia. This result suggested that odorant-stimulated PKC activity is mediated by the calcium-sensitive PKC beta isotype. Taken together, these results are consistent with the conclusion that PKC beta and beta ARK mediate odorant receptor phosphorylation and olfactory signal termination.

DNA sequence analysis supports the association of phytoplasmas with papaya (Carica papaya) dieback, yellow crinkle and mosaic

Previous studies using the polymerase chain reaction (PCR) with primers that hybridise to regions within and adjacent to the 16S rRNA gene have indicated that Australian papaya dieback, papaya mosaic and papaya yellow crinkle are associated with phytoplasmas (formerly called mycoplasma-like organisms). In this study, DNA sequence analysis of PCR products obtained from affected plant tissue has confirmed that the amplified DNA products are most similar to phytoplasmas associated with other plant diseases. These results support the proposition that phytoplasmas may be the causal agents of these economically significant papaya diseases in Australia. Further, a PCR product obtained from DNA extracts of Rutherglen bugs (Nysius vinitor) found on papaya plants, also had a sequence that was similar to those of other phytoplasmas. However, this sequence was different from any of the PCR products derived from diseased papaya. N. vinitor is an unlikely vector of phytoplasmas because of its mechanism of feeding, but this result suggests that it should be considered in future studies of the vector of papaya dieback, mosaic and yellow crinkle.

The dual expression of minor and major bcr/abl chimeric mRNA in blast crisis of chronic myelogenous leukemia

The hypothesis that minor bcr/abl fusion mRNA is produced in blast crisis of chronic myelogenous leukemia (CML) is examined. The RNA transcripts encoding the minor and major bcr/abl fused protein were detected by polymerase chain reaction (PCR) using RNA from peripheral blood or bone marrow cells of eight patients with blast crisis or accelerated phase of CML. The mRNA encoding for major bcr/abl was detected in all eight cases. In four patients, however, transcripts encoding for minor bcr/abl mRNA were detected, as well as major bcr/abl mRNA. The presence of minor bcr/abl mRNA was verified with the hybridization with a junction-specific probe and DNA sequencing analysis of PCR products. The appearance of minor bcr/abl fusion mRNA was associated with the lymphoblastic immunophenotype of the blast cells. In two of these four patients, samples of initial diagnosis of chronic phase of CML were available, which did not show minor bcr/abl transcript. We conclude that the appearance of minor bcr/abl mRNA transcript is associated with the terminal evolution of CML in lymphoblastic crisis.

Mutations and immunohistochemistry of p53 and proliferation markers in astrocytic tumors of childhood.

Thirty cases of hemispheric astrocytic tumors of childhood, consisting of 11 pilocytic astrocytomas, 2 fibrillary astrocytomas, 9 anaplastic astrocytomas, and 8 glioblastomas, were studied for the presence of p53 mutations and for immunohistochemical demonstrations of p53 and proliferation markers PCNA and Ki-67 MIB-1. The study was performed using polymerase chain reaction (PCR)-assisted single-strand conformation polymorphism analysis of exons 5-8 and direct sequence analysis of PCR products. For immunohistochemistry, DO1 and PAb 1801 were used. No mutation and no positivity for p53 protein were found in pilocytic astrocytomas. Mutations (at codons 144, 202, and 245) were found in 2 out of 8 glioblastomas and in 1 out of 9 anaplastic astrocytomas, whereas positive staining was found in 11 out of 17 malignant gliomas. Cases with mutations showed the highest p53 labeling index and also PCNA and MIB-1 labeling indices. The negative results in pilocytic astrocytomas are in line with the benign course of these tumors, whereas for malignant gliomas no difference seems to exist in comparison with adult cases.

Instability of human TATA-binding protein CAG triplet repeats during amplification by PCR

Polymerase chain reaction (PCR) of a TATA-binding protein cDNA that contains CAG triplet repeats results in heterogeneous products. This is caused by a variable loss in the number of CAG triplets. Sequence analysis of PCR products suggests that instability increases with repeat length.

Mutations of Ki-ras oncogene codon 12 in betel quid chewing-related human oral squamous cell carcinoma in Taiwan.

In Taiwan, there are two million people who have a betel quid chewing habit, and approximately 80% of all oral cancer deaths are associated with this habit. To investigate the incidence and types of Ki-ras codon 12 mutations in oral cancer associated with betel quid chewing, we used a sensitive mutation-specific two-stage polymerase chain reaction (PCR) technique to examine human oral squamous cell carcinomas from formalin-fixed, paraffin-embedded tissues. DNA sequence analysis of PCR products revealed that 6 of 33 (18%) tumour specimens contained Ki-ras codon 12 mutations. Four of the tumours contained more than one mutation. Three different base changes were detected, resulting from a substitution of wild type glycine (GGT) to either serine (AGT), aspartic acid (GAT) or cysteine (TAT). These results indicate that Ki-ras oncogene activation may play a role in the oncogenesis of betel quid chewing-related human oral squamous cell carcinomas.

Multiple mitochondrial DNA deletions in an elderly human individual

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Intact Alzheimer amyloid precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA

Using antibodies directed against N-terminal and C-terminal epitopes we have immunologically detected APP species in the membrane and saline-soluble fractions of unstimulated platelets, and in the conditioned medium of thrombin-stimulated platelets. These studies demonstrate an intact 140 kD membrane-associated form of APP that is released on degranulation. Evidence that platelets synthesize at least one form of APP (APP751) was obtained by enzymatic amplification of specific mRNA using Polymerase Chain Reaction (PCR) and direct sequence analysis of PCR product. Processing of APP for release may occur via successive C-terminal truncations, and/or by the release and proteolysis of an intact membrane associated form. An intact form of APP in platelets provides a circulating substrate upon which proteases from many tissues may act to produce beta protein (AB) during pathologic conditions.

Ras oncogene mutation in multiple myeloma.

The frequency of ras (H-, K-, and N-ras) and c-myc oncogenes was investigated in multiple myeloma (MM). By means of the polymerase chain reaction (PCR)/oligonucleotide hybridization method, DNA from 56 tumor biopsies was analyzed for the presence of activating mutations involving codons 12 and 61 of the H-, K-, and N-ras genes and codon 13 of the N-ras gene. Mutations, involving the N- or K-ras genes, were detected in 18 of 56 (32%) cases of which 12/43 (27%) were at diagnosis and 6/13 (46%) were after treatment. In some cases, multiple mutations affecting different ras alleles were detected. Direct nucleotide sequence analysis of PCR products indicated that a more heterogeneous nature of the base pair changes than previously shown for other tumors along with a preferential involvement of N-ras codon 61. The heterogeneity of MM cases with respect to the presence of ras oncogenes prompted an analysis of possible correlations with different clinico-pathologic characteristics of MM from which a correlation between the presence of ras oncogenes and a partial or complete lack of response to therapy emerged. The frequency of activating rearrangements or mutations of the c-myc gene were studied by Southern blot analysis and PCR sequencing, respectively. However, contrary to previous reports involving mostly MM cell lines, no structural alterations of the c-myc gene were found. These results indicate that ras, but not c-myc, oncogenes are activated in vivo in MM cells, representing the first oncogene alteration that has been associated at appreciable frequency with this type of malignancy. While the mechanism of occurrence and biological role of ras activation in MM remains to be elucidated, the preliminary correlations observed in this study between the presence of ras oncogenes and poor therapeutic response suggest that further investigations of the possible prognostic significance of these alterations are necessary.