Radioactive cDNA Probes Research Articles

Ileal Bile Acid-binding Protein, Functionally Associated with the Farnesoid X Receptor or the Ileal Bile Acid Transporter, Regulates Bile Acid Activity in the Small Intestine

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Haemagglutination as a confirmatory test for Peste des petits ruminants diagnosis

Seven Peste des petits ruminants (PPR) viruses and a RPV (vaccine strain) and their respective antiserum raised in rabbits were used for reciprocal cross neutralization test and haemagglutination (HA) and haemagglutination inhibition (HI) test employing 0.5% chicken RBC. Although many differential diagnostic methods for PPR and RP (rinderpest) are available viz., immunocapture ELISA, ‘N’ gene radioactive cDNA probes, non radioactively labelled biotinylated cDNA probes, reverse transcription-polymerase chain reaction (RT-PCR) for ‘F’ gene along with reciprocal cross neutralization test, HA and HI tests could be used as a reliable alternative in field conditions particularly in places where RP and PPR are co-existing. The advantages of the tests have been discussed in comparison with reciprocal cross neutralization tests.

βIi-tubulin and GAP 43 mRNA expression in chronically injured neurons of the red nucleus after a second spinal cord injury

Regeneration by chronically injured supraspinal neurons is enhanced by treatment of a spinal cord lesion site with a variety of neurotrophic and growth factors. The removal of scar tissue, with subsequent reinjury of the spinal cord, is necessary for injured axons to access tissue transplants placed into the lesion to support axon regrowth. The present study examined chronically injured and reinjured rubrospinal tract (RST) neurons to determine if changes in gene expression could explain the failure of these neurons to regenerate without exogenous trophic factor support. Adult female rats were subjected to a right full hemisection lesion via aspiration of the cervical level 3 spinal cord. Using radioactive cDNA probes and in situ hybridization, RST neurons in the contralateral red nucleus were examined for changes in mRNA levels of βII-tubulin and GAP 43 in an acute injury period (6 h–3 days), a chronic injury period (28 days after spinal cord injury (SCI)) and following a second lesion of the chronic injury site (6 h–7 days). Based upon the analysis of gene expression in single cells, GAP-43 mRNA levels were increased as early as 1 day following the initial SCI, but were no different than uninjured control levels at 28 days postoperative (dpo). The response to relesion was more rapid and higher than that detected after the initial injury with a significant increase in GAP 43 mRNA at 6 h that was maintained for at least 7 days. βII-tubulin mRNA levels remained unchanged until 3 days after an acute injury followed by a decrease in expression to 30% below uninjured control values at 28 dpo. The expression of βII-tubulin mRNA was significantly higher within 6 h after a second injury, where it remained stable for 5 days before a second increase occurred at 7 days after reinjury of the spinal cord. Thus, neurons in a chronic injury state retain the ability to respond to a traumatic injury and, in fact, neurons subjected to a second injury exhibit a significantly heightened expression of regeneration-associated genes.

Area and lamina-specific expression of GABAAreceptor subunit mRNAs in monkey cerebral cortex

Seven monkey-specific cDNAs corresponding to alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 2 GABAA receptor subunits were isolated and cloned; radioactive cDNA and cRNA probes were used for Northern blot analysis and in situ hybridization histochemistry of the primary visual, somatosensory, motor, temporal, and anterior parietal areas. alpha 1, beta 2, and gamma 2 subunit mRNAs were expressed at much higher levels than the other mRNAs, indicating that most cortical GABAA receptors are assembled from these three subunits. alpha 1, beta 2, and gamma 2 were also more highly expressed in area 17 than in all other areas, reflecting the greater density of GABA cells and synapses in area 17. In all cortical areas, each subunit mRNA showed an individual pattern of laminar expression. alpha 1, beta 2, and gamma 2 subunit mRNAs were particularly high in layers II-III, IV, and VI, tending to follow patterns of receptor binding and immunocytochemical staining. The other transcripts had different patterns, which did not match binding or immunocytochemical localization patterns. alpha 5 subunit mRNAs, which are highly expressed in development, were enriched in layer VI and the underlying white matter of visual cortex and in a layer IV-like strip in area 4, possibly reflecting the involvement of receptors formed from alpha 5 polypeptides in trophic interactions in the cortical subplate and in the transient layer IV during development of these areas. Monocular deprivation for 1-3 weeks, induced by intravitreal injection of tetrodotoxin, resulted in substantial reductions in levels of alpha 1, beta 2, and gamma 2 subunit mRNAs in deprived ocular dominance columns of the visual cortex, but the other subunit mRNAs were largely unaffected by monocular deprivation. In cortical layers in which expression of any of the transcripts was high, all neurons appeared to express the gene, but in layers in which expression was low or moderate, differences in the degree of labeling of individual neurons suggested that some neurons may not express certain subunit transcripts in detectable amounts. Laminar differences in expression of different GABAA receptor subunits in cerebral cortex suggest the assembly of functional receptors from different arrangements of available subunits in different types of cell. Receptors with different functional properties may be assembled from different combinations of subunit polypeptides in different layers and in the visual cortex may be differentially regulated under activity-dependent conditions.

Area and lamina-specific expression of GABA<SUB>A</SUB> receptor subunit mRNAs in monkey cerebral cortex

Seven monkey-specific cDNAs corresponding to alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 2 GABAA receptor subunits were isolated and cloned; radioactive cDNA and cRNA probes were used for Northern blot analysis and in situ hybridization histochemistry of the primary visual, somatosensory, motor, temporal, and anterior parietal areas. alpha 1, beta 2, and gamma 2 subunit mRNAs were expressed at much higher levels than the other mRNAs, indicating that most cortical GABAA receptors are assembled from these three subunits. alpha 1, beta 2, and gamma 2 were also more highly expressed in area 17 than in all other areas, reflecting the greater density of GABA cells and synapses in area 17. In all cortical areas, each subunit mRNA showed an individual pattern of laminar expression. alpha 1, beta 2, and gamma 2 subunit mRNAs were particularly high in layers II-III, IV, and VI, tending to follow patterns of receptor binding and immunocytochemical staining. The other transcripts had different patterns, which did not match binding or immunocytochemical localization patterns. alpha 5 subunit mRNAs, which are highly expressed in development, were enriched in layer VI and the underlying white matter of visual cortex and in a layer IV-like strip in area 4, possibly reflecting the involvement of receptors formed from alpha 5 polypeptides in trophic interactions in the cortical subplate and in the transient layer IV during development of these areas. Monocular deprivation for 1-3 weeks, induced by intravitreal injection of tetrodotoxin, resulted in substantial reductions in levels of alpha 1, beta 2, and gamma 2 subunit mRNAs in deprived ocular dominance columns of the visual cortex, but the other subunit mRNAs were largely unaffected by monocular deprivation. In cortical layers in which expression of any of the transcripts was high, all neurons appeared to express the gene, but in layers in which expression was low or moderate, differences in the degree of labeling of individual neurons suggested that some neurons may not express certain subunit transcripts in detectable amounts. Laminar differences in expression of different GABAA receptor subunits in cerebral cortex suggest the assembly of functional receptors from different arrangements of available subunits in different types of cell. Receptors with different functional properties may be assembled from different combinations of subunit polypeptides in different layers and in the visual cortex may be differentially regulated under activity-dependent conditions.

Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep.

Two experiments were conducted simultaneously to investigate regulation of amounts of mRNA for GnRH receptors during the periovulatory period in sheep. In the first experiment, amounts of mRNA for GnRH receptors were measured before and after preovulatory surge of LH following regression of the CL by prostaglandin F2 alpha(PGF2 alpha). So that the time of the preovulatory surge of LH could be accurately predicted, ewes received two injections of PGF2 alpha on Day 14 of the estrous cycle. Anterior pituitary glands were collected from 5 control ewes on Day 14 of the estrous cycle (0 h after PGF2 alpha) and at 48, 72, and 96 h after PGF2 alpha (5 ewes per group). The second experiment was conducted to investigate the effects of 17 beta-estradiol on amounts of mRNA for GnRH receptors. On Day 14 of the estrous cycle, 20 ewes were ovariectomized (OVX); 15 of these ewes received estradiol implants when they were OVX (OVXEI). Sixteen hours after OVX, anterior pituitary glands were collected from 5 OVX and 5 OVXEI ewes, and the remaining OVXEI ewes received an i.m. injection of estradiol (25 micrograms in corn oil; OVXEI + E) to induce a preovulatory-like surge of LH. Anterior pituitary glands were collected from OVXEI + E ewes 18 or 54 h after injection of estradiol (n = 5 per group). Half of each anterior pituitary gland was used to measure the number of GnRH receptors. Poly(A)+ RNA was isolated from the remaining half of each anterior pituitary gland, applied to slot blots, and hybridized with a radioactive cDNA probe encoding the ovine GnRH receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Lamina-specific expression and activity-dependent regulation of seven GABAA receptor subunit mRNAs in monkey visual cortex

Seven monkey-specific GABAA receptor subunit cDNAs were isolated and cloned; radioactive cDNA and cRNA probes derived from them were used for Northern blot analysis and in situ hybridization histochemistry of the primary visual cortex (area 17), with comparative observations on other cortical areas. cDNAs corresponding to alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 2 GABAA receptor subunits were isolated and had sequences unique to the monkey but recognized mRNAs of distinct molecular weights consistent with those reported in other species. mRNAs for the alpha 1, beta 2, and gamma 2 subunits were expressed at much higher levels in area 17 than in motor, somatosensory, or temporal association cortex, possibly reflecting the greater density of GABA cells and synapses in area 17. In areas 17 and 18, each of the seven subunit mRNAs showed individually distinct patterns of laminar distribution. alpha 1, beta 2, and gamma 2 subunit mRNAs, which are thought to form the basis of receptors with the full range of classical GABAA receptor properties in the adult, were particularly enriched in layers II-III, IVC, and VI of area 17, following patterns of receptor distribution previously demonstrated by radioligand binding and immunocytochemistry. alpha 2, alpha 4, alpha 5, and beta 1 transcripts had quite different localization patterns that did not match the antoradiographic or immunocytochemical receptor localization patterns. alpha 2 and alpha 5 subunit mRNAs, which are thought to be the subunits mainly expressed in development, were enriched in layer VI and the underlying white matter, possibly reflecting the involvement of receptors formed from alpha 2 and alpha 5 polypeptides in trophic interactions in the cortical subplate zone during development of the cerebral cortex. Following 8-21 d periods of monocular deprivation induced by intravitreal injection of TTX, levels of alpha 1, beta 2, and gamma 2 subunit mRNAs were substantially reduced in deprived ocular dominance columns of layer IVC in area 17. The effect was greatest for the alpha 1 subunit; for both alpha 1 and gamma 2 subunit mRNAs, it extended into deprived rows of cytochrome oxidase-identified periodicities in other layers. Apart from the alpha 5 subunit mRNA, which showed reduced levels in layer VI, the other subunit mRNAs were unaffected by monocular deprivation. These results demonstrate the heterogeneity of GABAA receptor subunit expression in a complex, multilaminar cortical area. They suggest that receptors with different functional properties may be assembled from different combinations of subunit polypeptides in different layers and show that subunit expression is differentially regulated under activity-dependent conditions.

Enhanced gene expression of calcium regulatory proteins in stunned porcine myocardium

Increasing evidence points to a molecular disturbance of Ca2+ homeostasis in stunned myocardium. The aim of this study was therefore to investigate the expression of mRNAs for Ca2+ binding proteins related to the sarcoplasmic reticulum in a porcine model of myocardial stunning. In 22 anaesthetised pigs, stunning was achieved by one or two cycles of 10 min left anterior descending coronary artery occlusion and reperfusion. Hearts were excised at various timepoints of the protocol. Total RNA was extracted from stunned (experimental) as well as normally perfused (control) myocardium. Northern blot analysis using radioactive cDNA probes revealed that the Ca(2+)-ATPase mRNA levels increased 1.6-fold compared to the control value at 90 min of the second reperfusion. The steady state level of phospholamban mRNA rose 2.5-fold at 180 min of reperfusion. A 2.3-fold increase in calsequestrin mRNAs was observed after 90 min of the second reperfusion. The calmodulin and alpha, beta myosin heavy chain mRNA levels were unchanged. A glyceraldehyde-3-phosphate dehydrogenase cDNA probe served as a reference system. Nuclear run-on assays showed increased transcription for Ca(2+)-ATPase and calsequestrin at 90 min of reperfusion, supporting the view that increased mRNA levels seen with northern hybridisation were due to increased transcription of the respective gene. The results suggest specific repair mechanisms of stunned myocardium and point to the involvement of calcium regulatory proteins related to the sarcoplasmic reticulum in the pathogenesis of myocardial stunning.

Isolation of a novel cDNA clone showing marked similarity to ME491/CD63 superfamily.

A novel cDNA clone, A15, was isolated by the differential screening of a cDNA library of an immature T cell line, HPB-ALL using radioactive cDNA probes from the mRNA of either HPB-ALL or peripheral blood lymphocytes. It hybridized to a single mRNA species of about 2.0 kilobases which is expressed in HPB-ALL cell line, but not in the PBL or a promyelocytic leukemia cell line, HL-60. The A15 gene codes for a protein of 244 amino acids which contains four potential transmembrane domains and four possible N-linked glycosylation sites. A computer-aided comparison showed a marked similarity to several other membrane proteins: CD9, CD37, CD53, TAPA-1, Sm23, CO-029, and ME491/CD63.

The gene for human growth hormone-releasing factor (GHRF) maps to or near chromosome 20p12

Growth hormone-releasing factor (GHRF), a hypothalamic releasing factor also named somatocrinin, influences the secretion and synthesis of growth hormone. Human GHRF is encoded by a single gene which was assigned to chromosome 20 by dot-blot analysis of DNA from dual laser sorted chromosomes. Using a radioactive cDNA probe, we localized the GHRF gene to chromosome 20p12 or near band 20p12.

Expression of prolactin gene in incubating hens

Steady-state levels of the 1.38-kb mRNA encoding the prolactin prohormone, measured by northern and dot blotting using a radioactive cDNA probe, rose 10-fold with concomitant increases in concentrations of prolactin in anterior pituitary and plasma from non-incubating to incubating stages in Gifujidori bantam hens. Daily injections of a low dose of thyrotrophin-releasing hormone (TRH, 0.25 micrograms/kg) for 4 days caused no changes in the concentrations of prolactin of the mRNA, but a high dose (2.5 micrograms/kg) of TRH did induce a significant increase in prolactin mRNA levels compared to saline controls. The results suggest that gene expression for prolactin increases substantially during the incubation period and TRH may contribute, at least in part, to the increase in transcription of prolactin mRNA for prolactin biosynthesis in Gifujidori bantam hens.

In situ hybridization with cDNA probes: Its application to the detection of mRNAs for myelin proteins (PLP and MBP) in mouse brain sections.

In situ hybridization is a method for identifying specific DNA or RNA sequences in cells or tissues. It was originally developed for the detection of specific DNA sequences in chromosomes (2, 6), then extended to the detection of specific endogenous mRNA in cells (5). Various kinds of nucleotide sequences, such as complementary DNA (cDNA), antisense RNA and synthetic oligonucleotide that have been labeled with radioactive or otherwise detectable molecules, could be acceptable as probes for in situ RNA hybridization, as well as Northern hybridization. Among these probes, the radioactive cDNA probe is the most widely used for in situ hybridization. With a rapidly increasing number of cDNAs corresponding to molecules of interest, in situ hybridization has been extensively used in various fields such as neurobiology, endocrinology, developmental biology, etc. The detection of mRNAs for myelin basic protein (MBP) and myelin proteolipid protein (PLP) (7, 12, 13) is a good example of successful application of in situ hybridization. Here we describe our evaluation of some technical conditions which are generally used in in situ hybridization. We also show the distribution of MBP and PLP mRNAs in the mouse brain sections revealed by the relatively simple but effective method of in situ hybridization.

Evaluation of a radioactive rRNA:cDNA-hybridisation assay for the direct detection of Chlamydia trachomatis in urogenital specimens.

A radioactive cDNA probe complementary to chlamydial ribosomal RNA was used to detect C trachomatis in urogenital specimens. Of 37 specimens positive with cell culture 31 were confirmed by the rRNA:cDNA hybridisation test, the sensitivity being 83.8%. The specificity of the hybridisation test was 94.4%, as 186 of 197 specimens that were negative by cell culture were also negative when assessed by the hybridisation method. Given a prevalence of 15.8% the predictive values for positive and negative results were 73.8% and 96.9%, respectively. In additional experiments the possible role of microorganisms added to the specimen collection medium was investigated. However, no indication for crosshybridisation was found; at high concentrations microorganisms interfered with the test procedure.

Duchenne muscular dystrophy: detection of deletion carriers by spectrophotometric densitometry.

DNA isolated from a family segregating a deletion in the Duchenne muscular dystrophy gene and control families was digested with restriction enzymes, Southern transferred, and probed with a radioactive dystrophin cDNA probe. The resulting autoradiographs were analyzed with a densitometric spectrophotometer to detect carriers of the deletion. The carrier status of females in the deletion pedigree was independently determined by genomic probes and confirmed by densitometry. In many Duchenne families, deletions will only be observed using cDNA probes which show few restriction fragment length polymorphisms (RFLPs). Such deletions would normally have to be detected using dosage gels. The spectrophotometric densitometry technique used by us does not require dosage gels, and avoids problems arising from non-informative meioses and cross-overs. It should be possible to screen every family with an exon deletion by spectrophotometric densitometry provided the presently available cDNA is suitably reduced to produce fewer bands on autoradiographs.

Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus

cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 × SSC, whereas 20 × SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30–50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.

Increased peroxisomal enzyme mRNA levels in adult rat hepatocytes cultured in a chemically defined medium and treated with nafenopin

Nafenopin is a known inducer of peroxisome proliferation in the hepatocytes of treated rodents. Primary cultures of adult rat hepatocytes maintained in a chemically-defined medium respond to the drug. RNAs from hepatocyte cultures treated for 1, 8 and 20 hr and their untreated counterparts have been purified and hybridized to radioactive cDNA probes specific for peroxisomal mRNAs (for catalase and the three enzymes of the β-oxidation system). The amount of the specific mRNAs was fairly constant or increased slightly in control cultures, but increased steadily during treatment of the cultures with a non-toxic dose of nafenopin (32 μ m). For the peroxisomal bifunctional enzyme mRNA, representative of the β-oxidation system, this increase was approximately fivefold after 20 hr, whereas for catalase mRNA a twofold increase compared with the control was observed after 20 hr. The time-course of the induction of the peroxisomal bifunctional enzyme mRNA in vitro was found to be similar to that observed after intragastric treatment of rats with nafenopin. This indicates that mechanistic studies on the early events induced in hepatocytes by peroxisome proliferators can be performed with this culture system. Such studies may help to explain the hepatotoxic/hepatocarcinogenic properties of this class of xenobiotics.

Complexes of viroids with histones and other proteins.

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.

Localization of the alpha and beta casein genes to the q24 region of chromosome 12 in the rabbit (Oryctolagus cuniculus L.) by in situ hybridization.

The syntenic alpha and beta casein genes were localized in the rabbit by chromosomal in situ hybridization, using a mixture of two radioactive cDNA probes corresponding to these two genes. Highly significant labeling was observed on chromosome 12. A total of 175 silver grains was found on chromosomes in the 193 mitoses studied; 18% of the grains were on chromosome 12, and 42% of the grains on this chromosome were localized to the 12q24 region. Statistical analysis revealed that this labeling was highly significant.

Winter flounder antifreeze proteins: a multigene family.

The nucleotide sequence of a cDNA clone of winter flounder antifreeze protein was determined by the dideoxynucleotide method. The sequence would predict a protein of 91 amino acids composed of a prepropeptide of 38 amino acids and a mature protein of 53 amino acids, which includes four complete 11-amino acid repeats. This predicted sequence corresponds to an antifreeze protein of intermediate size which is one 11-amino acid repeat longer than the smallest antifreeze proteins found in the serum of winter flounder during the cold season. Southern blot hybridization analysis of winter flounder genomic DNA with radioactive cDNA probes reveals a multigene family of potential antifreeze protein genes. This conclusion is supported by amino acid sequence analysis of several serum antifreeze proteins.

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating a alpha cells and asporogenous alpha alpha cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the a alpha sporulation probe relative to the alpha alpha control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, a alpha, and alpha alpha cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in a alpha cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in a alpha cells in sporulation medium.