Regional chromosomal assignment of human 3-beta-hydroxy-5-ene steroid dehydrogenase to 1p13.1 by non-isotopic in situ hybridisation

Summary In situ hybridisation using a biotinylated 1.2-kb human cDNA clone for human 3-beta-hydroxy-5-ene steroid dehydrogenase (HSD) supports the provisional regional localisation of the HSD gene to chromosome 1p13 and refines this localisation to 1p13.1.     

Computer assisted signal co-localization for simultaneous detection of antigen by immunohistochemistry and DNA by non-isotopic in situ hybridization

A method has been developed to co-localize signals for antigen and DNA using a desktop microcomputer system (computer assisted signal co-localization). Antigens were detected by standard immunohistochemical methods and DNA was detected by non-isotopic in situ hybridization (NISH). Using this method, NISH signals can be precisely located in cells with well-preserved morphology captured by computer. The removal of the first immunohistochemical reaction products and reagents eliminates possible interference with hybridization and non-specific binding to the probe; therefore the sensitivity of the original NISH method remains. The captured NISH signals can be converted to any other colour which contrasts with the immunostaining. We have used detection of Epstein-Barr virus (EBV) and keratins as a model system. This method is straightforward, and with necessary modifications, will be applicable to any type of combined immunohistochemistry and in situ hybridization technique for simultaneous detection of antigen and nucleic acids or two types of nucleic acids in the same cells.     

Oligo-riboprobes. Tools for in situ hybridisation

In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. Moreover, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes ("oligo-riboprobes"). These probes can be labelled to very high (10(9) cpm/micrograms) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise beta (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from beta preprotachykinin cDNA.     

Fluorescence in situ hybridisation studies provide evidence for somatic mosaicism in de novo dystrophin gene deletions

The fluorescence in situ hybridisation (FISH) technique was tested for its ability to detect somatic mosaicism in mothers of isolated deletion cases of Duchenne/ Becker muscular dystrophy. A control female with known germline and somatic mosaicism was examined, and both the normal cell line and the carrier cell line were detected. Subsequent FISH analysis of three other mothers of boys with apparent de novo dystrophin gene deletions revealed a second patient with a high level of somatic mosaicism, suggesting that a proportion of de novo dystrophin gene deletions occur as mitotic errors early in development rather than as meiotic errors during gametogenesis.     

Chromosomal localization of the ovine beta-casein gene by non-isotopic in situ hybridization and R-banding.

The ovine beta-casein gene (CNS2) has been mapped to a specific chromosome band using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe pTZ-E4 was a fragment of the ovine beta-casein gene inserted in the plasmid pTZ18R and labeled with biotin-11-dUTP. It hybridized to band q32 of ovine chromosome 4. The discrepancy between this result and the previous localization of this gene on cattle chromosome 6 may be explained by the very great similarity of the banding patterns of ovine and bovine chromosomes 4 and 6.     

The identification of microorganisms by fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported.     

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

OA1 mutations and deletions in X-linked ocular albinism.

X-linked ocular albinism (OA1), Nettleship-Falls type, is characterized by decreased ocular pigmentation, foveal hypoplasia, nystagmus, photodysphoria, and reduced visual acuity. Affected males usually demonstrate melanin macroglobules on skin biopsy. We now report results of deletion and mutation screening of the full-length OA1 gene in 29 unrelated North American and Australian X-linked ocular albinism (OA) probands, including five with additional, nonocular phenotypic abnormalities (Schnur et al. 1994). We detected 13 intragenic gene deletions, including 3 of exon 1, 2 of exon 2, 2 of exon 4, and 6 others, which span exons 2-8. Eight new missense mutations were identified, which cluster within exons 1, 2, 3, and 6 in conserved and/or putative transmembrane domains of the protein. There was also a splice acceptor-site mutation, a nonsense mutation, a single base deletion, and a previously reported 17-bp exon 1 deletion. All patients with nonocular phenotypic abnormalities had detectable mutations. In summary, 26 (approximately 90%) of 29 probands had detectable alterations of OA1, thus confirming that OA1 is the major locus for X-linked OA.     

Carrier detection in a partially dominant X-linked disease: ornithine transcarbamylase deficiency

Summary Ornithine transcarbamylase (OTC) deficiency is an X-linked disease responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes (15%). Increased orotic acid excretion occurs in both symptomatic and asymptomatic carriers, especially under protein loading tests. The disease is therefore partially dominant with neonatal lethality in the hemizygous male; the fraction of new mutations has previously been estimated to be low in males (point estimation = 0, upper bound of the confidence interval = 0.16) and 57% in females. Genetic counseling in this disease is difficult because it is not clear whether a negative protein loading test rules out carrier status. In an attempt to determine how reliable the test is for carrier detection, we investigated ten obligate carriers for orotic acid excretion; considering all data available, we concluded that the test is rarely negative in obligate carriers (8%). Consequently, a negative test in a mother decreases the minimum risk of being a carrier from 84% a priori to 30% if she had an affected son and from 43% a priori to 5% if she had a heterozygous daughter. Finally, the diagnosis of a new mutation in the germ cells of the maternal grandfather in one particular family could be ascertained by extensive DNA analysis.     

Sensitive detection of human growth hormone mRNA in routinely formalin-fixed,paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization

A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.     

Estimation of male to female ratio of mutation rates from carrier-detection tests in X-linked disorders.

A method is presented for the estimation of the ratio of male to female mutation rates from female carrier-detection test data from pedigrees containing an isolated male manifesting an X-linked necessive disorder. Pedigrees of any size and complexity (barring consanguinity) and containing any number of tested females can be utilized. The relative fitness of affected males and carrier females, and the segregation probability of the abnormal gamete in females, can be estimated simultaneously with the ratio of mutation rates in order to test specific hypotheses against given bodies of data. Here this method is applied to families containing isolated individuals affected with Lesch-Nyhan syndrome.     

Human papillomaviruses in anogenital warts in children: typing by in situ hybridisation.

OBJECTIVE--To identify the types of human papillomaviruses found in anogenital warts in children and to relate these to clinical and social information. DESIGN--In situ hybridisation using biotin labelled DNA probes to 11 types of human papillomavirus was performed on biopsy specimens from 17 children with anogenital warts. SETTING--Nuffield department of pathology and the department of dermatology, Oxford. PATIENTS--Children in one group were referred by general practitioners or paediatricians to the dermatology department, where biopsies were performed. The other children were seen in four different hospitals, and biopsy specimens were submitted to the laboratory at the physician''s or pathologist''s request. RESULTS--Of the 17 biopsy specimens, 10 contained cells positive with a probe to a genital human papillomavirus type (types 6 or 11), while six were positive with a skin virus type (types 2 or 3). One was negative. The virus type present bore no relation to the site or appearance of the warts. The virus type did, however, appear to correlate with groups of children. Skin types were commoner in older children (over 4 years), in those with a relative who had skin warts, and in children with warts elsewhere; there was no relation with the child''s sex and no suspicion of sexual abuse in these children. These circumstances suggested non-sexual transmission, such as autoinoculation. In contrast, genital types were commoner in girls, in children under 3 years, in children with relatives with genital warts, and in those with no warts elsewhere. Nevertheless, there was suspicion or evidence of sexual abuse in only half these children, suggesting that other routes of transmission--for example, perinatal--might have been implicated. CONCLUSION--Anogenital warts in children may contain either skin or genital wart virus type. Although the type of human papillomavirus present may give some indication of the likely mode of transmission, this can be interpreted only in conjunction with all available clinical and social information. The type of virus does not provide proof of the presence or absence of sexual transmission.     

Tissue distribution of bovine cystatin C analysed by in situ hybridisation

Cysteine proteinase inhibitors of the cystatin superfamily have been identified in many living organisms. However, knowledge of the tissue distribution of such inhibitors is limited. To elucidate this distribution in mammals, we have investigated the expression of the gene for cystatin C, belonging to cystatin family II, in several bovine tissues. In situ hybridisation with a digoxigenin-labelled cRNA probe demonstrated a high concentration of bovine cystatin C mRNA in the secretory epithelial cells of the choroid plexus, and also intense staining in cells of lymphoid tissue and in Sertoli cells. Cystatin C mRNA was also present in scattered neurons and glial cells throughout the cerebrum and the cerebellum. In the submandibular gland, specific mRNA was found mainly in striated intralobular ducts and interlobular ducts. The expression of cystatin C in brain tissue is of particular interest, as the inhibitor appears to be involved in certain neurological diseases. The main production of cystatin C within the brain is believed to be by astrocytes. However, this work shows that also neurons from young, normal individuals express cystatin C.     

Identification of the rice D-genome chromosomes by genomic in situ hybridisation

 The 24 rice D-genome chromosomes were identified among the 48 chromosomes of O. latifolia, which comprise the C- and D-genomes, using genomic in situ hybridisation (GISH). The B-genome chromosomes were also discriminated from the C-genome chromosomes in O. minuta (BBCC) by GISH. A comparison of the differences in the fluorescence intensity between the C and D genomes within O. latifolia (CCDD), and between the B and C genomes within O. minuta, indicated that the overall nucleotide-sequence homology between the B and C genomes is less than that between the C and D genomes. The origin of the D genome and the phylogenetic relationship of the D genome among the rice genomes are discussed, based on the results obtained. Received: 5 June 1997 / Accepted: 19 June 1997     

Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes.

A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.