DNA Oligonucleotide Probes Research Articles

Detection ofDesulfobacter in oil field environments by non-radioactive DNA probes

Acetate-utilizing, sulfate-reducing bacteria of the genusDesulfobacter were enriched from an oilfield seawater-injection system. These bacteria were detected in enrichment culture and verified asDesulfobacter by an oligonucleotide DNA probe that targets aDesulfobacter-specific sequence of ribosomal RNA. TheseDesulfobacter produced extensive biofilm in culture and exhibited high levels of hydrogenase activity, which suggests a sessile habit and a role in the cathodic depolarization mechanism of microbially influenced corrosion. Phospholipid analyses indicated that one of the oilfield isolates ofDesulfobacter lacked the fatty acid 10 Me 16:0, which has been used as a biomarker for this genus. Desulfobacter were infrequently encountered in the oilfield systems studied, and they constituted a small portion of the total microflora at any sampling location.

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.

Comparison of the Jβ gene usage among different T cell receptor Vβ families in spleens of C57BL/6 mice

To clarify whether there is a bias in the V-D-J combination of T cell receptor (TcR) genes, J beta gene usage has been investigated in a total of 743 TcR beta genes of V beta 2, V beta 8.2, and V beta 14 families expressed in C57BL/6 mouse spleens. Genes of TcR beta chains, amplified by a reverse transcriptase-polymerase chain reaction, were individually cloned into plasmids. Cloned genes (61 to 106), randomly selected in each respective V beta family from three different mice, were tested by means of hybridization with 12 oligo DNA probes which were designed to differentiate 12 murine functional J beta gene segments. The results are enumerated below. (1) The J beta 2.6 gene segment was found to be most frequently used (V beta 2, 19.8%; V beta 8.2, 21.2%; and V beta 14, 19.2%). In contrast, usage of the J beta 1.6 gene segment was most infrequent (V beta 2, 1.9%; V beta 8.2, 2.9%; and V beta 14, 0.5%); (2) High frequency of the J beta 2.1 gene segment and low frequency of the J beta 1.3 and J beta 1.5 gene segments were also observed; (3) The J beta 2 cluster was used in preference to the J beta 1 cluster (usages of the J beta 2 cluster: V beta 2, 67.8%; V beta 8.2, 65.9%; and V beta 14, 70.4%); and (4) These biases were generally common to all three V beta families examined and differences among individual mice were mostly small. Considering these findings, we conclude that the TcR J beta gene segments in C57BL/6 mice splenocytes are selected with a bias, but are selected independently of the V beta families.

Physical map of mouse chromosome 17 in the region relevant for positional cloning of the Hybrid sterility 1 gene.

Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

Induction of Interferon-γ, Interleukin-4, and Transforming Growth Factor-β in Rats Orally Tolerized against Experimental Autoimmune Myasthenia Gravis

Oral administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with Torpedo AChR + complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG) and the suppression of AChR-specific B cell responses and counteracts the development of AChR-reactive interferon-γ (IFN-γ) secreting T cells. To study the involvement of the T helper type 1 (Th1) cell-related lymphokine IFN-γ, the Th2 cell-related interleukin-4 (IL-4), and transforming growth factor β (TGF-β) that suppresses the synthesis of IFN-γ and IL-4, we used in situ hybridization with complementary DNA oligonucleotide probes to enumerate mononuclear cells (MNC) expressing mRNA for the cytokines IFN-γ IL-4, and TGF-β. Upon in vivo recognition of AChR, popliteal, inguinal, and mesenteric lymph nodes, spleen and thymus of rats with EAMG contained higher levels of IFN-γ, IL-4, and TGF-β mRNA-expressing cells compared to CFA-injected control rats, implicating the involvement in EAMG of AChR-reactive Th1 and Th2 cells in parallel. TGF-β was also upregulated in EAMG. Oral tolerance to EAMG was characterized by suppression of the levels of MNC expressing IFN-γ and IL-4, but augmentation of cells expressing TGF-β. The results suggest that IFN-γ, IL-4, and TGF-β are involved in the development of EAMG, and that TGF-β is important in the induction of oral tolerance to EAMG.

Increased transforming growth factor-beta, interleukin-4, and interferon-gamma in multiple sclerosis.

The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell-associated interferon-gamma (IFN-gamma), the Th2 cell-related interleukin-4 (IL-4), and the immune response-downregulating cytokine transforming growth factor-beta (TGF-beta), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN-gamma IL-4, and TGF-beta, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-beta- and IFN-gamma-positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-gamma and TGF-beta and TGF-beta mRNA expressing blood MNC but lower numbers of IL-4-positive cells. No or slight disability of MS was associated with high levels of TGF-beta mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-gamma-positive cells. IFN-gamma and TGF-beta may have opposing effects in MS, and treatments inhibiting IFN-gamma and/or promoting TGF-beta might ameliorate MS.

Detection of interferon gamma mRNA in the mucosa of patients with coeliac disease by in situ hybridisation.

In situ hybridisation has been used to study interferon gamma (IFN gamma) mRNA expression in the small intestine of patients with coeliac disease. Sections of jejunal biopsies were obtained from five patients with treated and five with untreated coeliac disease and five disease controls. These sections were hybridised with radiolabelled specific DNA oligonucleotide probes. The lamina propria of untreated coeliac disease patients contained a significantly increased number of IFN gamma producing cells compared with controls but there was no significant difference between the coeliac patients treated with a gluten free diet and controls. The results suggest that IFN gamma may play a part in the immunopathogenesis of coeliac disease.

Augmented interferon-γ, interleukin-4 and transforming growth factor-β mRNA expression in blood mononuclear cells in myasthenia gravis

The abnormal T lymphocyte-dependent production of antibodies to the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG) suggests a role for immunoregulatory cytokines. We examined the T helper type 1 (Th1) cell-associated interferon-γ (IFN-γ) that promotes cell-mediated immunity, the Th2 cell-related interleukin-4 (IL-4) that augments B cell immunity, and transforming growth factor-β (TGF-β) that downregulates immune responses but enhances isotype switching. Blood mononuclear cells (MNC) expressing cytokine mRNA were enumerated after in situ hybridization with labelled complementary DNA oligonucleotide probes for IFN-γ, IL-4 and TGF-β. MG patients had elevated numbers of cells expressing IFN-γ and IL-4 compared to patients with non-inflammatory neurological diseases and healthy controls, implying that both Th1- and Th2-like cells are involved in MG. TGF-β-positive cells were also elevated in MG. The levels of cytokine-positive MNC were similar in MG and in control patients with other inflammatory neurological diseases. There were no associations between numbers of cytokine-positive blood MNC and clinical variables of MG, but individual patients need to be studied over the course of MG to clarify a relation between the cytokines under study and clinical or laboratory variables of MG.

A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR TM) performed at 42°C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.

Organ-specific autoantigens induce interferon-gamma and interleukin-4 mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis.

T cells recognizing the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) are increased in multiple sclerosis (MS), and there are elevated numbers of T cells recognizing the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG). However, the cytokine repertoires in these diseases are largely unknown. We adopted in situ hybridization with radiolabeled complementary DNA oligonucleotide probes to enumerate mononuclear cells that expressed the T-helper type 1 (Th1) cell-related interferon-gamma (IFN-gamma) and Th2-associated interleukin-4 (IL-4) after short-term culture in the presence of autoantigen. High numbers of IFN-gamma and IL-4 mRNA-expressing cells in response to MBP and PLP were detected in patients with untreated MS, and to AChR in MG. The levels of IFN-gamma and IL-4 mRNA-positive cells in MS after culture in the presence of AChR, and in MG after culture in the presence of MBP or PLP, did not differ from those detected after culture without antigen. The CSF of MS patients contained four- to eightfold more myelin protein-reactive IFN-gamma and IL-4 expressing cells. The findings imply that MS and MG are associated with mixed Th1- and Th2-like cell responses directed to organ-specific target antigens.

Evidence for plant ribosomal 5S RNA involvement in elongation of polypeptide chain biosynthesis.

A series of short oligo-DNA probes (8-10-mers) complementary to various regions of the plant ribosomal 5S ribonucleic acid (5S rRNA) have been synthesized. The results of their hybridization to free 5S rRNA and to ribosomes pointed to the availability of nucleotides in loop "C" for complexation. We found a correlation between hybridization of selected oligonucleotides and their inhibitory effect on enzymatic binding of Phe-tRNA and poly(Phe) synthesis on wheat germ 80S ribosomes. Evidence was obtained for involvement of 5S rRNA in the elongation of polypeptide chain during protein biosynthesis. 5S rRNA seems to play a critical role in protein biosynthesis, probably through causing conformational changes of loop C.

HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity.

Cervical carcinoma is now known to be associated with human papillomaviruses (HPV), but the evidence for a link with specific HLA loci is controversial. The role of genetic variation at the HLA class II loci and among HPV types in cervical carcinoma was investigated by PCR DNA amplification and oligonucleotide probe typing of paraffin-embedded invasive cervical cancer tissue from Hispanic patients and of cervical swabs from Hispanic controls. Certain HLA class II haplotypes (such as DRB1*1501-DQB1*0602) were associated significantly, while DR13 haplotypes were negatively associated with cervical carcinoma. These associations are HPV16-type specific. These results suggest that specific HLA class II haplotypes may influence the immune response to specific HPV-encoded epitopes and affect the risk of cervical neoplasia.

Organ-specific autoantigens induce transforming growth factor-beta mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis.

Multiple sclerosis (MS) is characterized by patchy accumulations of inflammatory cells combined with demyelination. There are mononuclear cells in blood and cerebrospinal fluid of patients with MS that produce interferon-gamma and interleukin-4 in response to myelin basic protein (MBP) and proteolipid protein (PLP). Here we describe autoantigen-induced production of transforming growth factor-beta (TGF-beta). This multifunctional cytokine has inhibitory effects on the growth, differentiation, and effector functions of activated T cells. Blood and cerebrospinal fluid cells were exposed in short-term cultures to MBP and PLP and, after hybridization with complementary DNA oligonucleotide probes, they were evaluated for TGF-beta mRNA expression. Patients with MS had higher numbers of MBP- and PLP-responsive TGF-beta mRNA expressing cells in blood compared with control patients with other neurological diseases or myasthenia gravis and a five- and threefold further increment in their cerebrospinal fluid. In blood of patients with myasthenia gravis, where the acetylcholine receptor (AChR) is a target for autoaggressive immunity, there were increased levels of AChR-responsive TGF-beta mRNA expressing cells. Thymectomized myasthenia gravis patients showed higher levels of TGF-beta mRNA expressing cells compared with patients not thymectomized. Numbers of cells responding to AChR in MS and MBP in myasthenia gravis did not differ from numbers found in absence of antigen. Patients with other neurological diseases showed infrequent and low responses to MBP, PLP, and AChR. Diseases with presumed autoimmune pathogenesis are associated with organ-specific autoantigen-induced TGF-beta production, which is increased after thymectomy.

In Situ Hybridization for Ribosomal RNA Sequences: A Rapid Sensitive Method for Diagnosis of Infectious Pathogens in Anatomic Pathology Substrates.

Ribosomal RNA (rRNA) sequences are present in all prokaryotic and eukaryotic cells. These sequences are highly conserved among organisms and are know to be species specific. Sequence analysis is widely utilized in diagnostic microbiology in order to identify infectious agents. A rapid in situ hybridization assay for detection of rRNA sequences in formalin or Bouin's fixed paraffin embedded tissues was developed using biotin-labeled oligonucleotide DNA probes specific for a variety of rRNA sequences. This method was used effectively to identify human, Aspergillus species, Pneumocystis carinii, and Toxoplasma gondii rRNA sequences in a protocol requiring less than 30min to complete. Utilization of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.

Optic neuritis is associated with myelin basic protein and proteolipid protein reactive cells producing interferon-γ, interleukin-4 and transforming growth factor-β

Studies on patients with monosymptomatic optic neuritis (ON) should give opportunities to identify features typical for early multiple sclerosis (MS). There are increased T and B cell responses to the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) in both ON and MS, but there is little information on the types of cytokines produced by such cells. We describe the use of in situ hybridization with complementary DNA oligonucleotide probes to detect and enumerate mononuclear cells expressing mRNA for the cytokines interferon-γ (IFN-γ) which augments cell-mediated immunity; interleukin-4 (IL-4) which promotes the B cell response; and transforming growth factor β (TGF-β) that in many cases downregulates immune responses. Expression of these cytokines was studied in mononuclear cells from peripheral blood and cerebrospinal fluid (CSF) from patients with ON and MS after in vitro exposure to MBP and PLP, and in absence of antigen. There were elevated levels of cells that in response to MBP and PLP expressed IFN-γ, IL-4 and TGF-β mRNA in blood and further enriched in CSF in both ON and MS, compared to patients with other neurological diseases. The results suggest that IFN-γ, IL-4 as well as TGF-β are involved in both ON and MS, and that the cytokine profile in early MS as reflected by ON is not different from that in clinically definite MS.

Opioid peptide gene expression in the nucleus trctus solitarius of rat brain and increases induced by unilateral cervical vagotomy: Implications for role of opioid neurons in respiratory control mechanisms

Neurons expressing messenger RNA encoding the opioid peptide precursors, preproenkephalin and preprodynorphin were localized in the medulla oblongata of the rat by in situ hybridization of specific DNA oligonucleotide probes. Neurons containing preproenkephalin messenger RNA were found throughout the medullary reticular formation in the gigantocellular and paragigantocellular reticular nuclei, the parvicellular and lateral reticular nuclei; commissural, medial and ventrolateral subnuclei in the nucleus tractus solitarius and the nucleus of the spinal trigeminal tract. Labelled cells were also concentrated in the more medial regions of the area postrema. In contrast, neurons containing preprodynorphin messenger RNA had a more restricted distribution and were detected in the commissural and ventrolateral nucleus tractus solitarius and nucleus of the spinal trigeminal tract, especially in the more dorsal regions. Expression of preproenkephalin and preprodynorphin messenger RNA was also examined in the dorsal vagal complex of rats that had undergone a unilateral nodose ganglionectomy or cervical vagotomy. Twenty-four hours after both cervical vagotomy and nodose ganglionectomy, there was a specific 1.5–2-fold elevation in preproenkephalin and preprodynorphin messenger RNA levels in the ventrolateral subnucleus of the contralateral nucleus tractus solitarius relative to levels in the ipsilateral nucleus tractus solitarius and in the nucleus tractus solitarius of sham-operated animals. Previous immunohistochemical studies demonstrating the co-localization of enkephalin and dynorphin in the ventrolateral nucleus tractus solitarius suggest that these changes occurred in the same population of neurons. In light of the suggested role of the ventrolateral nucleus tractus solitarius as a central respiratory centre and the activation of the intact pulmonary afferents that innervate this area following a unilateral vagotomy (which increases inspiration volume and expiratory time by affecting the Hering-Breuer reflex), our results suggest a specific involvement of enkephalin- and dynorphin-containing neurons in the ventrolateral nucleus tractus solitarius in central respiratory control mechanisms.

An analysis with sequence-specific oligonucleotide probes of the association between aspirin-induced asthma and antigens of the HLA system

Background: Previous work has suggested that in addition to environmental influences, there is a genetic predisposition for the development of both asthma and atopy. A subset of asthmatic subjects who are intolerant to aspirin has been identified. A previous study has suggested that there is an association between aspirin sensitivity and HLA-DQw2. Methods: To further assess this association, we studied two populations of aspirin-sensitive subjects and aspirin-tolerant subjects with asthma. Genomic DNA was amplified by polymerase chain reaction and hybridized with radiolabeled oligonucleotide probes specific to the second exon of the DRB1, DQA1, DQB1, and DPB1 chains. Results: Using polymerase chain reaction amplification of genomic DNA and sequence-specific oligonucleotide probes, we have been unable to confirm a significant association between aspirin sensitivity and HLA-DQw2. Conclusion: We have shown, however, that there is a significant decrease in the incidence of DPB1 *0401 in both aspirin-tolerant and aspirin-intolerant subjects with asthma in both populations studied.

Use of digoxigenin-labelled oligonucleotide DNA probes for VT2 and VT2 human variant genes to differentiate Vero cytotoxin-producing Escherichia coli strains of serogroup O157

Digoxigenin-labelled oligonucleotide DNA probes specific for B-subunit genes of Vero cytotoxin 2 (VT2) and a variant of VT2 (VT2vha) were used to differentiate 116 strains of Escherichia coli serogroup O157 belonging to phage types 1, 2, 4, 8, 14, and 49. Of these strains, 38% had sequences for both VT2 and VT2vha, 38% had sequences for VT2 only, and 24% had sequences for VT2vha only. Oligonucleotide probe hybridization subdivided strains of all of the phage types except phage type 1. The greatest variation in toxin gene pattern was observed with strains of phage type 14, for which there were six distinct patterns when the presence or absence of VT1 genes was also considered. Two strains from each phage type group were examined for bacteriophages encoding VT production. Two of the six VT2vha-producing strains carried phage from which DNA hybridized with the VT2vha-specific probe. Phages were not detected in the remaining four VT2vha strains, suggesting that genes may be chromosomally located or associated with a defective prophage. In contrast, seven of the eight VT2 strains carried phages from which DNA hybridized with the VT2-specific probe. Two strains of E32511 (O157:H-) were also investigated. One strain (E32511A) possessed gene sequences for both VT2 and VTvha and was shown to carry phage possessing gene sequences for VT2. With strain E32511B, however, phages were not detected and DNA hybridized only with the VT2vha probe. Analysis of total genomic DNA digested with restriction endonuclease EcoRI showed that polymorphisms were seen with VT2 strains and not with VT2vha strains.

Localization of hepatitis C viral RNA and capsid protein in human liver

In the livers of patients whose sera contained antibodies to C100-3 antigen (anti-HCV) and hepatitis C virus (HCV) RNA, the presence of HCV RNA and HCV capsid protein (CP) antigen was demonstrated by in situ hybridization and immunohistochemistry, respectively. It was found that occasional hepatocytes in four of ten livers from patients whose sera were positive for both anti-HCV and HCV RNA hybridized with antisense as well as sense oligonucleotide DNA probes, whereas the probes did not hybridize with livers from patients whose sera were negative for anti-HCV and HCV RNA. Monoclonal antibody against a synthetic oligopeptide with amino acid sequence of HCV CP reacted with occasional hepatocytes in six of 14 livers from patients whose sera contained these HCV markers, but not with livers from patients whose sera were negative for both of them. These results suggest that HCV proliferates within hepatocytes since both antisense and sense probes hybridized with cytoplasm of the hepatocytes and that the virus matures in the cytoplasm as the capsid proteins were also found in the hepatocytes.

Age-related changes in expression of the neural cell adhesion molecule in skeletal muscle: a comparative study of newborn, adult and aged rats

Neural cell adhesion molecule (NCAM) is expressed by muscle and involved in muscle-neuron and muscle-muscle cell interactions. The expression in muscle is regulated during myogenesis and by the state of innervation. In aged muscle, both neurogenic and myogenic degenerative processes occur. We here report quantitative and qualitative changes in NCAM protein and mRNA forms during aging in normal rat skeletal muscle. Determination of the amount of NCAM by e.l.i.s.a. showed that the level decreased from perinatal to adult age, followed by a considerable increase in 24-month-old rat muscle. Thus NCAM concentration in aged muscle was sixfold higher than in young adult muscle. In contrast with previous reports, NCAM polypeptides of 200, 145, 125 and 120 kDa were observed by immunoblotting throughout postnatal development and aging, the relative proportions of the individual NCAM polypeptides remaining virtually unchanged at all ages examined. However, changes in the extent of sialylation of NCAM were demonstrated. Even though the relative amounts of the various NCAM polypeptides were unchanged during aging, distinct changes in NCAM mRNA classes were observed. Three NCAM mRNA classes of 6.7, 5.2 and 2.9 kb were present in perinatal and young adult skeletal muscle, whereas only the 5.2 and 2.9 kb mRNA classes could be demonstrated in aged muscle. This indicates that metabolism of the various NCAM polypeptides is individually regulated during aging. Alternative splicing of NCAM mRNA in skeletal muscle was studied by Northern blotting using DNA oligonucleotide probes specifically hybridizing to selected exons or exon combinations. Exon VASE, which has previously been shown to be present in both brain and heart NCAM mRNA, was virtually absent from skeletal muscle at all ages studied. In contrast, the majority of NCAM mRNA in postnatal skeletal muscle was shown to contain extra exons inserted between exons 12 and 13. Of the various possible exon combinations at this splice site, the combinations 12-a-AAG-13 and 12-a-b seemed to be prevalent in postnatal skeletal muscle. No significant change in the relative proportion of these two exon combinations occurred during aging. The observed upregulation of NCAM protein in aged muscle supports the assumption that an increasing proportion of muscle fibres are denervated in aged muscle. Selective upregulation of the 5.2 and 2.9 kb mRNA forms have previously been demonstrated in muscle cell lines and in primary cultures of muscle cells during formation of myotubes in vitro, and this switch in NCAM mRNA classes has been suggested to correlate with myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)