Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) offers polymerase chain reaction tests for an increasing range of single gene defects, and fluorescence in situ hybridization tests for sex determination (for X-linked conditions) and for aneuploidy detection. Patients carrying chromosome translocations with a high reproductive risk are increasingly seeking to increase their chances of a normal pregnancy with the help of PGD, for which they present a special challenge. This paper describes the behaviour of reciprocal translocations at meiosis, discusses current methods of detecting meiotic outcomes at the preimplantation stage and outlines ways forward for preimplantation diagnosis of these common rearrangements. We also propose a more general strategy using recently developed chromosome-specific sub-telomeric probes, combined, if possible, with proximal probes, to form a strong diagnostic tool.     

Detection of human papillomavirus in cervical scrapings by in situ hybridization and the polymerase chain reaction in relation to cytology

A gynaecological out-patient population consisting of 200 patients aged 19-43 years (mean age 34.2 years) was screened for the presence of human papillomavirus (HPV) by the polymerase chain reaction and in situ hybridization on cervical scrapings. A novel method was applied for the detection of HPV in cervical cells by embedding them in a paraffin block before in situ hybridization was performed. This technique resulted in well preserved cytological morphology, easy performance and economy of probes. In eight of the 200 patients (4%), human papillomavirus DNA was revealed by the polymerase chain reaction. Subtyping revealed the presence of HPV serotype 16 DNA in three of these patients. In one patient HPV serotype 18 DNA was also present. The in situ hybridization assay was able to detect all those cases with a specific HPV serotype infection.     

Recent developments in the understanding of antinuclear autoantibodies

Cytological identification of soybean mitotic metaphase chromosomes (2n = 40) has been severely limited by their small size and uniform karyomorphology. We have developed fluorescent in situ hybridization (FISH), PCR-primed in situ labelling (PCR-PRINS) procedures, and molecular probes for routine cytological identification and for the physical mapping of soybean somatic chromosomes. Chromosome preparation has been achieved by modifications of previous protocols and through the preparation of root-tip protoplasts prior to chromosome spreading. Initially our probe selection focused on highly repeated DNAs that provide very intense localized hybridization signals. Repetitive gene probes that have proven valuable include the rDNA loci (5S and 45S) which are chromosome specific. We have also developed satellite DNA probes for two different sequence families: the SB92 and the STR120 satellites. Both of these are tandemly arranged at multiple chromosomal loci. By using different cloned examples of each family, we have been able to selectively label unique subsets of soybean chromosomes. Double hybridization with biotin and digoxigenin labeled probes has allowed us to determine the chromosomal overlap between different probes. In addition, we have joined portions of the metaphase chromosome painting patterns with the genetic map by single-copy FISH and PCR-PRINS detection of the RFLP loci G8.15, G17.3, and A199a and A199b. Total genomic DNA in situ hybridization (GISH) patterns were also used to characterize the soybean chromosomes.     

Polymerase chain reaction-reverse cross-blot hybridization assay in the diagnosis of sporotrichoid Mycobacterium marinum infection

In this paper, we report a patient in whom Mycobacterium marinum sporotrichoid infection was diagnosed using polymerase chain reaction (PCR) amplification of the 16S rRNA gene and subsequent analysis of the amplified product in a reverse cross-blot hybridization assay with mycobacterial species-specific probes. This molecular method allowed us rapidly to detect and identify this organism directly in the patient's lesional skin biopsy rather than in cultures in conventional media. The identification provided by PCR-reverse cross-blot hybridization assay was confirmed by examination of the morphological and biochemical features and by high-performance liquid chromatography analysis of mycolic acid from the clinical isolate, suggesting the validity of our molecular approach.     

Susceptibility of human lymphoblastoid B-cell line CE to HIV1 infection

OBJECTIVE: To establish a simple, accurate and rapid method for screening of the mutant genes in phenylketonuria (PKU). METHODS: Four exons harboring the mutations, Y204C (exon6, E6), R243Q(E7), Y356X(E11) and R413P(E12), were amplified by polymerase chain reaction (PCR) with incorporation of biotinylated deoxynucleotide(biotinylated-11-dUTP or biotinylated-14-dCTP). Hybridization between immobilized allele-specific oligonucleotide probes and biotin-labelled amplified DNA was performed and nonradioactively detected by a colorimetric reaction using streptavidin-alkaline phosphatase. RESULTS: The methods of non-radioactive reverse dot blot hybridization were established to screen the mutations. We detected the genotypes of five PKU patients and found that three of them carried R243Q mutation and one of the three also carried Y356X mutation. These results were confirmed by PCR-single strand conformation polymorphism. CONCLUSION: This method is suitable for rapid screening for common mutations in Chinese PKU patients.     

Time-resolved fluorometric hybridization assays with RNA probes synthesized from polymerase chain reaction-generated DNA templates

DNA templates suitable for direct synthesis of RNA probes are produced by the polymerase chain reaction. The nucleic acid sequence of interest is amplified using a downstream primer carrying the T7 RNA polymerase promoter sequence. The modified primer is incorporated into the amplified DNA, which is subsequently used for RNA probe synthesis in the presence of T7 RNA polymerase and a hapten-labeled ribonucleotide (digoxigenin-UTP). As a model, we prepared RNA probes specific for the BCR-ABL mRNA characteristic of chronic myelogenous leukemia. The probes are used in time-resolved fluorometric hybridization assays. Mixtures of BCR-ABL positive and negative cells, as well as whole blood samples, are analyzed. The sample mRNA is amplified using a biotinylated upstream primer. The amplified product (target DNA) is captured onto streptavidin-coated wells and hybridized to the RNA probe. The hybrids are detected with an alkaline phosphatase (ALP)-labeled antibody. ALP hydrolyzes the phosphate ester of fluorosalicylic acid, and the fluorosalicylate produced forms highly fluorescent ternary complexes with Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. As low as 0.4 fmol of target DNA can be detected. Also, a single leukemic cell may be detected in the presence of 0.5 million "normal" cells.     

The presence of human papillomavirus in sinonasal papillomas, demonstrated by polymerase chain reaction with consensus primers

The frequency of human papillomavirus (HPV) in sinonasal papillomas seems to vary considerably. The highest frequencies have been reported by investigators using in situ DNA or RNA hybridization. Few studies have used polymerase chain reaction, and in these reports the frequency of HPV detection is rather low. We have investigated the presence of HPV in sinonasal papillomas using the polymerase chain reaction with a set of degenerated consensus primers, which amplify the vast majority of the known HPV types. Human papillomavirus was found in three of 14 papillomas. By in situ hybridization the same three papillomas were positive for HPV type 6/11.     

A PCR assay for detection of a 2RL.2BS wheat-rye chromosome translocation

A 2RL.2BS wheat-rye translocation, present in the wheat germplasm line Hamlet, carries a gene for resistance to Hessian fly biotype L, one of the most virulent biotypes presently encountered in wheat production environments. Unlike several other wheat-rye chromosome translocations common in wheat breeding programs, 2RL lacks genes encoding storage proteins or other easily selected markers. Oligonucleotide primers synthesized from published sequences derived from the R173 family of moderately repetitive rye DNA were used in the DNA polymerase chain reaction (PCR) to identify specific markers for 2RL. The same primers, when used with DNA extracted from additional wheat-rye translocation lines of importance to the wheat breeding community, gave distinctive PCR products for each genotype. The single primer pair, PAWS5 and PAWS6, may, therefore, have wide applicability for the identification of wheat-rye chromosomal translocations presently encountered in wheat breeding populations.     

DNA subtraction in situ hybridization--new approaches of detecting genetic defects in surgical pathology specimens

A novel in situ histochemical method of screening genetic alterations of human malignant cells was reported. The method is based on subtraction in situ hybridization of whole genomic DNA from a healthy volunteer or DNA extracted from non-neoplastic tissues. The method can widely examine the possible DNA defects in situ without employing specific probes employing colorimetric reaction in routinely processed surgical pathology materials, i.e. 10% formalin-fixed and paraffin embedded tissue sections. Results can be correlated with histopathological findings. In this report, we also describe the results on genetic alterations of human esophageal squamous cell carcinoma, using this DNA subtraction in situ hybridization.     

The expression of a NMDA receptor gene in guinea-pig myenteric plexus

Muscle tension studies of guinea-pig ileum longitudinal muscle-myenteric plexus preparations suggest that the N-methyl-D-aspartate (NMDA) receptor may be present in the enteric nervous system. Therefore, we investigated the expression of a gene for the NMDA receptor in guinea-pig taenia coli. The gene product was amplified using polymerase chain reaction (PCR) and its synthesis localized using in situ hybridization. A NMDA receptor PCR product from the myenteric plexus was demonstrated with nearly identical sequence characteristics to that in the brain. In situ hybridization studies identified myenteric neurons which express NMDA receptor messenger RNA. Demonstration of the genetic expression of the NMDA receptor supports a role for glutamate as a neurotransmitter in the enteric nervous system.     

Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping

Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.     

A revised strategy for cloning antibody gene fragments in bacteria

Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.     

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface

DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes. We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome. This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells. These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage P1, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences. Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains). In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis.     

Pilocarpine tablets for the treatment of dry mouth and dry eye symptoms in patients with Sj?gren syndrome: a randomized, placebo-controlled, fixed-dose, multicenter trial. P92-01 Study Group

The sequestration of RNA in Alzheimer's disease (AD) senile plaques (SPs) and the production of intraneuronal amyloid-beta peptides (Abeta) prompted analysis of the mRNA profile in single immunocytochemically identified SPs in sections of AD hippocampus. By using amplified RNA expression profiling, polymerase chain reaction, and in situ hybridization, we assessed the presence and abundance of 51 mRNAs that encode proteins implicated in the pathogenesis of AD. The mRNAs in SPs were compared with those in individual CA1 neurons and the surrounding neuropil of control subjects. The remarkable demonstration here, that neuronal mRNAs predominate in SPs, implies that these mRNAs are nonproteinaceous components of SPs, and, moreover, that mRNAs may interact with Abeta protein and that SPs form at sites where neurons degenerate in the AD brain.     

Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3

The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.