Oligo-riboprobes

Summary 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. More-over, 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 (10⁹ cpm/g) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from preprotachykinin cDNA.     

Technical evaluation of cDNA microarrays

The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

Sequence and expression of a type II keratin, K5, in human epidermal cells.

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.     

Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons

Rat genomic clones were used to quantitate preprotachykinin mRNAs in the rat basal ganglia, while the tachykinin peptide products substance P and substance K were measured by radioimmunoassay. Administration of the dopamine antagonist (antipsychotic) drug haloperidol significantly decreased substance P, substance K, and both alpha (substance P encoding) and beta (substance P/substance K encoding) preprotachykinin mRNAs, suggesting a drug-induced decrease in striatonigral tachykinin biosynthesis. The time course for decreased preprotachykinin mRNAs and tachykinins apparently parallels the period of maximum risk for the development of certain antipsychotic drug-induced extrapyramidal side effects seen clinically. Tachykinin interaction with dopamine neurons may play an important role in the modulation of basal ganglia function.     

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.     

Co-localization of LH beta and FSH beta mRNAS in the porcine anterior pituitary by in situ hybridization with biotinylated probes

The localizations of mRNAs encoding LH beta and FSH beta in porcine pituitary were investigated by in situ hybridization technique. Biotinylated porcine LH beta and FSH beta cDNA probes were used on frozen sections of paraformaldehyde-fixed pituitary specimens. Hybridizations to both mRNAs were observed specifically in cytoplasm with unstained nuclei. Furthermore, cells hybridized for LH beta mRNA were demonstrated to be identical to those for FSH beta mRNA. This study provided the first morphological evidence that both gonadotropin beta genes are expressed in the same cell.     

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.     

Sequence analysis of cloned cDNA for rat substance P precursor: existence of a third substance P precursor

The sequence of the mRNA for the rat substance P precursor (preprotachykinin A) has been elucidated by molecular cloning and sequence analysis. The deduced amino acid sequence of rat preprotachykinin A indicates that it contains both substance P and substance K but differs in the sequence organization from either bovine alpha- or beta-preprotachykinin A reported previously. The existence of the bovine mRNA for the third preprotachykinin A has thus been examined and evidenced by the isolation of the corresponding cDNA clone. This mRNA, named gamma-preprotachykinin A mRNA, deletes the sequence precisely corresponding to the exon 4 sequence of the preprotachykinin A gene. Thus, alternative RNA splicing in the expression of the single preprotachykinin A gene results in the generation of three different forms of the preprotachykinin A mRNAs.     

Triiodothyronine nuclear binding capacity in rat tissues correlates with a 6.0 kilobase (kb) and not a 2.6 kb messenger ribonucleic acid hybridization signal generated by a human c-erbA probe

Recent studies have raised the possibility of multiple structurally distinctive tissue-specific nuclear T3 receptors, all exhibiting homology with the v-erbA oncogene and represented by mRNAs of various sizes. We have assayed the level of mRNAs recognized by a 32P-labeled cRNA derived from human plancetal c-erbA-A beta cDNA by solution hybridization and by Northern transfer in different rat tissues, as well as human liver and placenta. Two related mRNAs were demonstrated in the rat tissues analyzed, one measuring 6.0 and the other 2.6 kilobases (kb). In human liver and placenta a 6.0 kb mRNA was seen, but not a 2.6 kb mRNA. Only the 6.0 kb sequence correlated with the receptor concentration determined by 125I-T3 displacement analysis.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Reduction in S100 protein beta subunit mRNA in C6 rat glioma cells following treatment with anti-microtubular drugs

S100 protein is a calcium-binding protein found in vertebrate nervous tissue. Synthesis of S100 protein in the rat glioma cell line, C6, is inhibited by the addition of anti-microtubular drugs. We have cloned a cDNA for the beta subunit of S100 protein from rat brain in a lambda gt 11 expression vector and used this cDNA to measure the amounts of S100 beta subunit mRNA in C6 cells after treatment with anti-microtubular drugs. Levels of alpha-tubulin and beta-actin mRNAs were also measured. All measurements were performed using RNA-RNA hybridization techniques at high stringency with rat mRNA-specific probes. After 24 h of treatment, the S100 beta subunit mRNA was reduced to levels of 25% by colchicine and 32% by vinblastine when compared to untreated controls. In contrast, the levels of tubulin and actin mRNAs were only slightly changed by these treatments. These studies demonstrate that disruption of the microtubular cytoskeleton causes a specific reduction in the level of S100 protein mRNA in C6 cells.     

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.