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Dual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host-pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT 25 -conjugated Oligo Affinity Support resin (dT 25 -OAS) alongside poly-dT 14 magnetic beads and dT 25 -cellulose. dT 25 -OAS was found to have the highest dA 21 oligo binding capacity at 4 pmol/µg, followed by dT 14 -magnetic beads (1.1 pmol/µg) and dT 25 -cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT 25 -cellulose showed the highest mRNA-affibody purification specificity, followed by dT 25 -OAS and dT 14 -magnetic beads. Overall, dT 25 -OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A) + mRNAs. To elute the poly(A) + mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A) + mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis)., (© 2019 Cold Spring Harbor Laboratory Press.)

Recent studies have shown that tissue-specific transcriptomes contain multiple types of RNAs that are transcribed from intronic and intergenic sequences. The current study presents a tool for the discovery of transcribed, unannotated sequence elements from RNA-seq libraries. This RNA Element (RE) discovery algorithm (REDa) was applied to a spectrum of tissues and cells representing germline, embryonic, and somatic tissues and examined as a function of differentiation through the first set of cell divisions of human development. This highlighted extensive transcription throughout the genome, yielding previously unidentified human spermatogenic RNAs. Both exonic and novel X-chromosome REs were subject to robust meiotic sex chromosome inactivation, although an extensive de-repression occurred in the post-meiotic stages of spermatogenesis. Surprisingly, 2.4% of the 10,395 X chromosome exonic REs were present in mature sperm. Transcribed genomic repetitive sequences, including simple centromeric repeats, HERVE and HSAT1, were also shown to be associated with RE expression during spermatogenesis. These results suggest that pervasive intergenic repetitive sequence expression during human spermatogenesis may play a role in regulating chromatin dynamics. Repetitive REs switching repeat classes during differentiation upon fertilization and embryonic genome activation was evident., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA-peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued. To overcome this problem, we have developed a low cost solid-phase synthesis protocol to generate oligo-dT cellulose. Comparative binding studies indicate that chemically synthesized oligo-dT cellulose functions with superior loading capacity when compared to the discontinued product. We suggest that this method could be used to synthesize oligo-dT resin for routine purification of poly-adenylated biomolecules., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry-compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation.

Background: Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. Here we document potent inhibition of real-time quantitative polymerase chain reaction (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis., Methods: We characterize DSS inhibition of qPCR in-vitro and in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize real-time polymerase chain reaction amplification and gene expression analysis., Results: DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly-A purification of DSS-exposed RNA allows reliable and cost-effective gene expression analysis in DSS-exposed tissue., Conclusions: DSS is a potent inhibitor of real-time qPCR amplification and interferes with tissue-specific gene expression analysis in DSS-exposed mice. Poly-A purification of tissue-derived RNA results in reliable and cost-effective gene expression analysis in DSS-exposed mice., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Early detection of colon cancer can result in a high cure rate; therefore, an accurate screening method is imperative. Adoption of non-invasive testing designed to reduce anxiety over colorectal cancer screening and improve early detection is highly desirable. Therefore, we have developed a novel non-invasive methodology utilizing exfoliated colonocytes in order to quantify colonic messenger RNAs (mRNAs). Previously we have demonstrated in the rat that intact eukaryotic mRNA can be isolated due to the presence of exfoliated colonocytes in the faecal stream. To assess use of this methodology in humans, this pilot study evaluated exfoliated colonocyte mRNA expression of 11 putative biomarkers using real-time reverse transcription-polymerase chain reaction (RT-PCR) in seven normal subjects, four subjects with inflammation, and 10 tumour-bearing subjects presenting for colonoscopy. Expression of the biomarkers was evaluated following normalization to TATA box binding protein mRNA levels. Tumour-bearing subjects diagnosed with adenoma had elevated levels of cyclin Dl (p = 0.041). In addition, subjects displaying inflammation of the colon exhibited higher mRNA levels of cyclooxygenase-2 (p = 0.007). These data suggest that mRNA isolated from exfoliated colonocytes could be used to detect early stages of colon cancer, and possibly chronic inflammation. To broaden the utility of non-invasive marker analysis, additional studies are needed to generate a multi-target assay panel of diagnostic markers. This will allow for the development of robust classifiers that can determine critical gene sets for the diagnosis and prediction of colon cancer in animal models and humans.

The present study investigated the effect of adenosine on glial glutamate efflux. Adenosine (from 1 nM to 100 microM) enhanced the release from cultured rat glial cells in a bell-shaped dose-responsive manner for the hippocampus and in a dose-dependent manner for the superior colliculus, and a similar increase was obtained with the A2a adenosine receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680), but not with the A1 adenosine receptor agonist, N6-cyclohexyladenosine (CHA). Adenosine and CGS21680 also enhanced glutamate efflux from Xenopus oocytes injected with the poly (A)+ mRNAs derived from cultured glial cells for the hippocampus and the superior colliculus together with and without the A2a adenosine receptor mRNA, but instead such increase was not found in oocytes expressing A2a adenosine receptors alone. The results of the present study thus suggest that adenosine enhances glutamate efflux from glial cells via A2a adenosine receptors, and this may represent a mechanism underlying the facilitatory action of adenosine on hippocampal and superior colliculus neurotransmissions.

A full-length complementary DNA (cDNA) clone of the hepatitis C virus (HCV) genome was used to prepare full-length plus- and minus-strand RNA. The minus-strand RNA, which contains a polyA(+) tract complementary to the polyU tract found in the plus strand (genomic) RNA, but not the plus strand RNA, was captured with a commercial polyA(+)-tract isolation system. After elution, the minus strand was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The combination of this procedure and RT-PCR using rTth resulted in an unprecedented level of discrimination of 10 logs(10). HCV minus-strand RNA isolation was unaffected by the addition of an excess of 10(4) of plus strands or by the addition of cellular RNA, and although the polyA(+) isolation step removed 99. 99% of plus strands, there was no loss of minus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples examined. Because the titer of plus-strand HCV RNA in any sample makes a significant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to confirm the replication of HCV in a population of cells.

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.

The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.

The conjugate between oligo(dT)16 and thermo-responsive polymer, poly(N-isopropylacrylamide), was prepared for isolation of poly(A)+ RNA from total RNA. The hybridization reaction between the conjugate and poly(A) (average length: 320 base) was equilibrated in 10 min, and all the poly(A) (16 nmol base for 24 nmol base of conjugate) was precipitated when raising the solution temperature to 35 degrees C. The precipitate was dissolved in water, and poly(A) was dissociated from the conjugate by heating to 65 degrees C. This separation system was successfully applied to the isolation of poly(A)+ RNA from total RNA.

A triblock self-associating polymer with the structure n-dodecane-poly(ethylene oxide)-n-dodecane and a very low polydispersity has been used as a matrix to separate a sample of single-stranded oligonucleotides containing Pd(A)25-30 and Pd(A)40-60. Above a concentration of 4%, this associative polymer forms a micellar network with cubic order and a well-defined micellar spacing, in which the dodecane micellar cores are bridged by polyoxyethylene segments. This medium combines a low viscosity with excellent resolution of oligonucleotides. This work confirms that associative polymers are potentially powerful media for separation in capillary electrophoresis, and argues in favor of the use of monodisperse products presenting a high-order in the physical gel state.

The linear chromosomal ends are usually unclonable by general methods due to a lack of restriction sites. We present a novel method, isolation of chromosomal ends (ICE), which has been developed for the efficient isolation of linear DNA ends.

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.

The interaction between polyadenylic acid (5') (poly [A]) and histone (or protamine) was analyzed by electrophoretic retardation of poly [A]-histone (or protamine) complex in agarose gel. The potency of interaction was protamine > histone H1, arginine-rich histone > other histones. The catalytic subunit of cyclic AMP-dependent protein kinase effectively decreased the electrophoretic retardation of poly [A]-histone H1. The interaction between poly [A] and histone H1 was also detected by the drastically enhanced absorbance around 340 nm. The findings may implicate a regulatory role of histone H1 on mRNAs through its binding on poly [A] tails.

The capillary electrophoretic separation of oligonucleotides (12-24 bases in length) using a micellar system or an entangled polymer solution was compared. Micellar electrokinetic capillary chromatography was performed using a fused-silica capillary whereas a J&W DB-17-coated capillary was used for capillary electrophoresis in entangled polymer solution. In both cases, a number of parameters were evaluated to improve separation. For example, the influence of concentration of copper(II) ions and sodium dodecyl sulfate, variation in applied voltage and temperature were investigated in the micellar system. The influence of variation in pH was also studied. The two methods were also compared with respect to their efficiencies. The results of quantitation showed better within-day and day-to-day repeatability for the entangled polymer solution. Both methods gave a comparable limit of detection of about 30 pg at a signal-to-noise ratio of 3.

Separation of various nucleic acids was evaluated by high-performance ion-exchange chromatography on non-porous resin, TSKgel DNA-NPR. A 1 kb ladder DNA was studied as a model DNA on operational variables like flow rate, gradient time, temperature, sample load, etc.. As results, various DNA fragments were well separated within 15 min by 20 min linear gradient at a flow rate between 0.5 and 0.75 ml/min at room temperature while the resolutin was dependent on molecular weight of the sample. The relationship between sample load and its peak area was examined on polymerase chain reaction (PCR) product. The product was found to be quantitatively recovered even with nanogram loads. The detection limit was 3.8 ng at signal to noise level (S/N) of 3. This non-porous ion-exchanger also showed high resolution on separation of ther nucleic acids like transfer RNA, oligonucleotides (single-stranded) DNA.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

We have isolated two cDNAs of 1.7 and 3.0 kb, produced by alternative splicing, that encode a angiotensin II (AII) receptor from a Xenopus laevis heart cDNA library. The two clones had identical coding regions with each other and were found to belong to the G protein-coupled receptor superfamily like the mammalian type 1 AII receptors (AT1); their amino acid sequence was 68.7% homologous with the human AT1 receptor sequence. However, there was a 1.3 kb insertion at the 3'-untranslated region of the longer clone. The insertion contained 9 repeats of an ATTTA motif, suggesting that the two mRNAs undergo distinct post-transcriptional regulation by virtue of a difference in their stability. Although the Xenopus receptor exhibited distinct specificities for AII receptor antagonists compared with mammalian AII receptors, several common characteristics, including the effect of dithiothreitol and guanosine 5'-O-(3-thiotriphosphate), demonstrated that the cloned receptor is a counterpart of the mammalian AT1 receptor. Moreover, the cloned receptor was expressed most abundantly in the Xenopus heart, which is inconsistent with the tissue distribution of mammalian AII receptors. This indicated that the Xenopus heart, unlike that of mammals, plays a major role in the AII-dependent regulation of blood pressure and extracellular fluid volume.

A cDNA clone (designated as GP2-7) encoding a novel 5-hydroxytryptamine (5-HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5-HT receptor subtypes (34-48%). The sequence of GP2-7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5-HT7. mRNA for GP2-7 was detected in cortical and limbic brain regions. Transiently expressed GP2-7 showed high-affinity binding to [3H]5-HT (pKi = 9.0) with the following rank order of affinities: 5-carboxyamidotryptamine (5-CT) > 5-HT = 5-methoxytryptamine (5-MeOT) > methiothepin > 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) > spiperone >> sumatriptan. Adenylyl cyclase activity in CHO-K1 cells transiently transfected with GP2-7 was stimulated by several analogues of 5-HT with the following order of potency: 5-CT > 5-HT = 5-MeOT > dipropyl-5-CT > 8-OH-DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2-7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5-CT.

Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.

7B2 is a 23-kDa protein encoded by a single gene that is expressed in a variety of neuroendocrine tissues. Although its physiological role has not yet been elucidated, its presence in secretory granules suggests a function in the secretory machinery of certain neuronal and endocrine cells in various species. The present study characterizes the expression of 7B2 in endocrine pancreatic cells. We demonstrate that: (i) 7B2 is highly expressed in human insulinomas; (ii) its ultrastructural localization, associated with secretory granules of A and B cells of the islets, suggests a participation of 7B2 in the secretion of insulin and glucagon; (iii) sequences located in the first intron of the 7B2 gene are required for its transcription in either insulinoma or glucagonoma cell lines; and (iv) in a B cell-like insulinoma cell line, the transcription of 7B2 is regulated by protein kinase A and protein kinase C activators, while in an A-like insulinoma cell line, 7B2 gene transcription seems to be constitutively activated.

Six connexin genes have previously been shown to be expressed in the rat kidney. Given the structural and functional diversity of the kidney, we hypothesize that other connexin genes may be expressed. We have partially screened a rat kidney cDNA library using low-stringency hybridization conditions with cDNA probes from rCx 43 and rCx 26 and report here the isolation of two connexin cDNA clones, rCx 30.3 and rCx 31, that have not previously been shown to be expressed in the rat kidney. Furthermore, rCx 30.3 utilizes two distinct transcripts in the kidney, while rCx 31 utilizes two transcripts in skin but only one in the kidney.

Inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of the 48-kDa regulatory protein arrestin. We recently isolated a novel form of arrestin, termed p44, that is truncated at the COOH terminus (Palczewski, K., Buczylko, J., Ohguro, H., Annan, R. S., Carr, S. A., Crabb, J. W., Kaplan, M. W., Johnson, R. S., and Walsh, K. A. (1994) Protein Sci. 3, 319-329) and strongly inhibits Gt activation by non-phosphorylated rhodopsin. p44 is identical to arrestin except at the COOH terminus, where the 35 amino acids of arrestin are replaced by a single alanine residue. p44 is identified as a splice variant of arrestin based on the identical cDNA sequence of p44 with arrestin (except the 3' non-coding regions), the presence of an exon/intron junction at the Ser369 codon, and identical Southern hybridization patterns generated by the 3' non-coding portion of arrestin and p44. Immunocytochemistry reveals that p44 is localized in the photoreceptor outer segment, whereas arrestin is present throughout the cell. This specificity of localization to the outer segment is consistent with a role of p44 in the phototransduction cascade.

Following a standard heat shock, approximately 40% of Hsp70 transcripts in Drosophila melanogaster lack a poly(A) tail. Since heat shock disrupts other aspects of RNA processing, this observation suggested that heat might disrupt polyadenylation as well. We find, however, that as the temperature is increased a larger fraction of Hsp70 RNA is polyadenylated. Poly(A)-deficient Hsp70 RNAs arise not from a failure in polyadenylation but from the rapid and selective removal of poly(A) from previously adenylated transcripts. Poly(A) removal is highly regulated: poly(A) is (i) removed much more rapidly from Hsp70 RNAs than from Hsp23 RNAs, (ii) removed more rapidly after mild heat shocks than after severe heat shocks, and (iii) removed more rapidly after a severe heat shock if cells have first been conditioned by a mild heat treatment. Poly(A) seems to be removed by simple deadenylation rather than by endonucleolytic cleavage 5' of the adenylation site. During recovery from heat shock, deadenylation is rapidly followed by degradation. In cells maintained at high temperatures, however, the two processes are uncoupled and Hsp70 RNAs are deadenylated without being degraded. These deadenylated mRNAs are translated with low efficiency. Deadenylation therefore allows Hsp70 synthesis to be repressed even when degradation of the mRNA is blocked. Poly(A) tail shortening appears to play a key role in regulating Hsp70 expression.

Pulmonate freshwater snails contain two different ferritin types, soma ferritin and yolk ferritin. A cDNA library was constructed from midgut gland poly(A)-rich RNA of the snail Lymnaea stagnalis L. and recombinant clones encoding both ferritin types were obtained by immunoscreening. The longest cDNA inserts had a length of 859 bp (soma ferritin) and 1548 bp (yolk ferritin) and the specificity of these inserts was confirmed by immunoprecipitation of both ferritin types translated in vitro from hybrid-selected mRNAs. The 5' untranslated region (UTR) of the soma ferritin mRNA contains a 28-bp element which shows 64% sequence identity with the iron-responsive element (IRE) of vertebrate ferritin mRNAs. The soma ferritin mRNA is strongly translated in the wheat germ system but poorly translated in rabbit reticulocyte lysate. The yolk ferritin mRNA, which contains no IRE, is equally well translated in both in vitro translation systems. The deduced amino acid sequence of the soma ferritin subunit (174 amino acid residues, M(r) 20140) shows 50-70% sequence identity with subunits of vertebrate ferritins. After removal of an 18-amino-acid-residue signal sequence the deduced protein sequence of yolk ferritin contains 221 amino acids (M(r) 25438). Sequence identity of this chain with other eukaryotic ferritin chains is only 31-42%. Both snail ferritin sequences are more similar to the H-subunit type of vertebrate ferritins than to the L-type and both have the H-specific amino acid residues of the ferroxidase centre. The yolk ferritin sequence has a 42-amino-acid-residue insertion predicted to reside in the L loop of the subunit.

Keratin 14 (K14) expression has recently been demonstrated in cell lines of non-parenchymal hepatic origin (Bisgaard et al., 1993, Mol. Carcinog., 7:60-66; Bisgaard et al., 1991, J. Cell. Physiol., 147:333-343). These cell lines are thought to represent a progeny of a dormant stem cell compartment present in the adult rat liver, which may participate in the restoration of the liver mass after experimental liver injury. Utilizing a combination of 2-acetylaminofluorene (2-AAF) administration and partial hepatectomy to activate liver regeneration by proliferation of oval cells, we examined the modulation of K14 as well as alpha-fetoprotein (AFP) expression in proliferating oval cells and lineages hypothesized to be derived herefrom. We showed by Northern blot and in situ hybridization analyses that K14 and AFP transcripts were initially accumulating in epithelial cells located in subsets of ductal structures in the portal areas. As oval cells infiltrated the liver parenchyma, K14 transcripts were detected in oval cells, in foci of small basophilic hepatocytes, and in structures resembling glandular intestinal-type epithelium. AFP was expressed in oval cells, and at low but detectable levels in foci of basophilic hepatocytes, but not in glandular intestinal-type epithelium. Neither K14 nor AFP transcripts were detected in bile ducts or mature hepatocytes at any time during oval cell proliferation and reconstitution of the liver mass. To further study the modulation of K14 and AFP expression we utilized an in vitro model in which spontaneous transformation of rat liver epithelial (RLE) cells appeared to mimic the process of early differentiation along the hepatic lineage in vivo. We demonstrated that undifferentiated RLE cells at a late passage expressed K14 and vimentin, whereas transformation and differentiation to hepatoblast-like progeny resulted in an abrogation of K14 and vimentin expression and an induction of K18 and AFP. We propose that K14 and AFP are sequentially modulated in subpopulations of oval cells involved in the ongoing reconstitution of the liver mass.

The role of cis-acting destabilizing RNA sequences in the determination of endocrine gene expression has been investigated using a novel paradigm, in which the differential regulation of two alternatively polyadenylated RNA transcripts may be observed both in vivo and in vitro. In the rat anterior pituitary gland in vivo, we have shown that, after the termination of an estrogen stimulus, a 1.7-kilobase (kb) vasoactive intestinal peptide (VIP) RNA containing an extensive 3'-untranslated region (UTR), is preferentially down-regulated with respect to a 1.0 kb VIP transcript that is uniquely abundant in this tissue. Differential regulation of the anterior pituitary VIP transcripts can be modeled in an explant culture system in which we defined both transcriptional and posttranscriptional phases of VIP gene regulation in vitro, and showed that selective down-regulation of the 1.7-kb transcript is posttranscriptional. Inhibitors of transcription and translation have also allowed us to show in vitro that differential regulation of VIP transcripts occurs through an active process that appears to involve the synthesis of a labile, destabilizing factor. In order to confirm the role of RNA destabilization as the primary mechanism of differential posttranscriptional regulation, we have also performed cell-free stability assays in which explant extracts were incubated with 32P-labeled run-off transcripts corresponding to the two alternatively polyadenylated VIP RNAs. The resultant estimates of RNA half-life showed significantly lower values for the synthetic VIP transcript containing the 3'-UTR. Our findings demonstrate the presence of functional destabilizing sequences in the 3'-UTR of the rat VIP RNA which appear to act in the physiological control of VIP gene expression.

Within the hippocampus, stimulus-transcriptional coupling plays an important role in post-seizure neuronal adaptation, post-ischemic cell death and the induction of long-term potentiation. To identify additional mediators of hippocampal transcriptional responses a targeted approach was developed and used to characterize the spectrum of nuclear hormone receptors expressed within this brain region. cDNAs encoding the DNA-binding domains of six different members of the nuclear hormone receptor superfamily were isolated. A majority were identical or closely related to receptors known to be expressed within the hippocampus. Two additional isolates, HZF-2 and HZF-3, encode the DNA-binding domain of novel members of the nuclear hormone receptor superfamily.

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

Kinesin is a microtubule-based motor protein involved in organelle transport in neuronal and nonneuronal cells. Although a single kinesin motor has been thought to serve all cell types, we document here that neurons express a second conventional kinesin heavy chain (nKHC) that is 65% identical in amino acid sequence to the ubiquitously expressed kinesin heavy chain (uKHC). By preparing antibodies which distinguish between the two KHCs, we demonstrate that nKHC is a nucleotide-dependent microtubule-binding protein which partially cofractionates with membrane organelles. Immunolocalization experiments show that nKHC is distributed throughout the CNS but is highly enriched in subsets of neurons. In hippocampal neurons in culture, uKHC is distributed uniformly throughout the neuron, whereas nKHC is selectively concentrated in the cell body. These results demonstrate that mammalian neuronal tissue contains two conventional kinesin motors which may serve distinct functions in microtubule-based transport.

The expression and cellular targeting of murine acetylcholinesterase (AChE) was examined after transient transfection of a human 293 cell line with a cDNA encoding the hydrophilic T-subunit. Expression of the recombinant clone produced catalytically active AChE either bound to the cell membranes, in an intracellular pool, or secreted into the medium. About 22% of the cell-associated AChE was membrane-linked as dimers and tetramers, required Triton X-100 for extraction, and bound to Triton X-100 as assessed by sucrose gradients. Immunocytochemical staining of live and permeabilized cells showed reactive epitopes at the plasma membrane. Assays of cell surface AChE activity indicated about 18% of the cellular enzyme was oriented on the external surface of the plasma membrane. Isotopic labeling of cultures with precursors of fatty acylation showed incorporation of [3H]palmitate into the membrane-bound fraction of AChE only. The label was sensitive to cleavage by mild alkaline methanol treatment, and the cleaved lipid was identified as methyl palmitate by thin layer chromatography, indicating covalent linkage of the fatty acid through an ester or thioester residue. Thus the membrane-bound AChE is palmitoylated, suggesting that fatty acylation may serve as an alternative mechanism for anchoring the hydrophilic polypeptide subunit of AChE to the external face of the plasma membrane.

Two cDNA fragments of terminal deoxynucleotidyl transferase, which contained new insertion sequences, were found from bovine thymus cDNA. One of the clones encoded 18 new amino acids and the other encoded 9 new amino acids. In the results of genomic structure analysis around the new insertion sequences, alternative splicing of the bovine terminal deoxynucleotidyl transferase gene was suggested.

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.

Malignant hyperthermia (MH) is an autosomal dominant myopathy. Molecular genetic studies have shown that the alteration of Arg615 to Cys in the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) is cosegregated with porcine MH (Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V. K., Weiler, J. E., O'Brien, P. J., and MacLennan, D. H. (1991) Science 253, 448-451; Otsu, K., Khanna, V. K., Archibald, A., and MacLennan, D. H. (1991) Genomics 11, 744-750). Here, using the fluorescence calcium indicator indo-1, we determined the concentration of ionized cytosolic calcium in myoblastic cells transfected with either the wild-type or mutated ryanodine receptor cDNA. The cells expressing the mutant ryanodine receptor showed higher sensitivity to caffeine, which induces Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor. Exposure to clinical doses of halothane resulted in a rapid increase of [Ca2+]i in cells expressing the mutated ryanodine receptor, whereas no [Ca2+] changes were observed in cells expressing the wild-type ryanodine receptor. These results provide definite evidence that a single amino acid mutation, Arg615-->Cys, in the ryanodine receptor is causative of MH.

A hippocampal 38 kd autoantigen recognized by an autoantibody from the serum of a patient with paraneoplastic limbic encephalitis (PLE) and small cell lung carcinoma (SCLC) was isolated by screening a human hippocampal cDNA library. The 1,991-nucleotide ple21 clone was obtained and the deduced 350-residue protein encoded by the ple21 cDNA clone was found to be highly homologous to the neuron-specific RNA recognition motifs (RRMs)-containing proteins. The homologies were confined to the RRMs and the RRM connecting region. The presence of RRM in the antigenic protein may be important in the pathogenesis of SCLC-associated paraneoplastic neurologic syndrome.

Polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of venom glands in Taiwan cobras to facilitate the cloning and sequencing of phospholipase A2 (PLA2) gene. The PCR product was then subcloned into pUC18 vector and transformed in E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method. Sequencing several clones containing about 0.5 kb DNA inserts constructed a complete and unambiguous full-length reading frame of 468 base pairs covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid segment of signal peptide. The sequenced major PLA2 with pI 4.991 shows a high degree of sequence homology to those PLA2 of the same or closely-related genus. The deduced protein sequence allows us to correct and resolve some discrepancy between the sequences determined by conventional protein sequencing (Toxicon, 19, 141(1981)) and X-ray crystallography (Science, 250, 1560(1990)). Expression of PLA2 in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified PLA2 from the same cobra venom albeit with a much lower activity.

RT PCR employing poly(A+)RNA from porcine luteal cells and a combination of primers designed from the known bovine MCP-1 cDNA identified the luteal cells as a source of MCP-1. This finding is corroborated by results from Northern analysis using total RNA from luteal cells. To characterize the complete porcine MCP-1 cDNA, poly (A+)RNA was isolated from porcine corpus luteum, transcribed into cDNA and the latter cloned into the expression vector lambda Uni-ZapXR. A digoxigenin-labeled DNA probe of 375 bp was obtained by PCR and employed to screen the library. From the positive clones pMCP5, pMCP7 and pMCP10, the clone pMCP5 was selected and both strands of the cDNA insert were sequenced. The cDNA insert was 742 bp long, with an open reading frame (ORF) encoding a protein of 99 amino acid residues which by comparison with known amino acid sequences of MCPs yielded highest identities with MCP-1 sequences. We therefore assume that pMCP5 encodes the amino acid sequence for porcine MCP-1.

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Three different clones have been isolated from a genomic library of the silkmoth Antheraea polyphemus by employing a subtractive hybridization technique. The clones with inserts of 13-16 kb of DNA each, code for mRNAs expressed in the wing epidermis during JH induced second pupal cuticle deposition. While two of the clones code for a single mRNA each, the third one codes for two mRNAs. All the four mRNAs code for distinct polypeptides that can be precipitated with antibodies raised against pupal cuticular proteins. These genes are activated at the same period of pupal development and their transcripts follow similar patterns of accumulation. Although these genes are expressed in a tissue and time specific manner attesting to their pupal wing epidermal specificity, three of them are expressed in the adult wing epidermis also, but not at the larval stage. While DNAs from other silkmoths and insects hybridize to these genes, only one of the A. polyphemus genes hybridizes to RNA from second pupal wings of two other silkmoths tested.

Previously, increased expression of mRNA encoding the p53 tumor suppressor protein was described during castration-induced regression of the rat ventral prostate gland with Northern blot techniques. This activity was confirmed with a ribonuclease protection assay that demonstrated a 16-fold induction of p53 transcripts in ventral prostate RNA within 72 hrs after castration. The induced expression of p53 mRNA correlated with increased detection of p53 protein in nuclei of regressing prostate epithelial cells. Immunohistochemical staining with anti-p53 antibody was strongly reactive for epithelial nuclei in castrated glands but unreactive for nuclei of control adult glands. In contrast to the upregulation of p53 in regressing prostate glands with a large proportion of apoptotic cells, expression of p53 mRNA was decreased in rat prostate glands that were stimulated to regrow by testosterone replacement.

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Frog (Rana catesbeiana) skeletal muscle (M-type) and liver (L-type) cDNAs of fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase were isolated from lambda gt10 phage cDNA library. The full-length L-type cDNA (1829 bp) encodes a 469 amino acids subunit (M(r) 54,800), while the M-type cDNA (1792 bp) encodes 455 amino acids (M(r) 52,901). The amino acid sequence of the M-type isozyme is identical to that of the L-type isozyme except for the N-terminus. The N-terminal 30 amino acids of the L-type isozyme are replaced by an unique sequence of 16 amino acids in the M-type isozyme. Both L- and M-type cDNAs were detected also in a lambda gt10 phage library of skeletal muscle. Relative amount of the M- and L-type mRNAs is skeletal muscle was determined by the reverse transcription-polymerase chain reaction method. The M/L mRNA ratio in frog skeletal muscle shows seasonal variations, being 0.56/1 in early summer and 5.3/1 in winter. These results suggest that there is a seasonal change in the isozyme composition and that the glycolysis in frog skeletal muscle may be regulated by type of the isozyme synthesized.

Capitalizing on the significant sequence homology comprising the transmembrane motif regions of known prostanoid receptor family, we targeted the cloning of a cDNA clone for prostaglandin (PG) F2 alpha receptor from a bovine corpus luteum cDNA library. By using several pairs of degenerated primers created from a common motif of transmembrane domains, polymerase chain reaction gave a clone SN463 carrying the homologous sequence, which covered transmembrane motif IV-VI of the thromboxane (TX) A2 receptor. This polymerase chain reaction product was used as a DNA probe for the following cross-hybridization, and a clone BC2211 carrying a 2.2-kilobase pair DNA insert was isolated. This clone encodes a protein of 362 amino acid residues (M(r) = 40,983) with seven potential transmembrane domains and represented significant overall sequence homology to human TXA2 receptor protein (34% in amino acid). Injection of the mRNA synthesized in vitro from the cloned cDNA into a Xenopus oocyte elicited electrophysiological response to PGF2 alpha. Ligand binding displacement in membranes of mammalian COS-7 cells transfected with the cDNA indicated the rank order of affinity of the receptor to PGs: PGF2 alpha > PGD2 > PGE2 > STA2, a TXA2 agonist. PGF2 alpha activated inositol phosphate formation in COS-7 cells transfected with receptor cDNA. Northern blot analysis and in situ hybridization indicated that the PGF2 alpha receptor mRNA is highly expressed and accumulated in corpus luteum. This is the first report on a successful cloning of functional receptor cDNA for PGF2 alpha.

The terminal stage of myogenic development is marked by the cessation of replication, fusion, and expression of genes which encode the myofibrillar proteins. Prior to terminal differentiation one or more stages of myogenic development take place. Expression of alpha 7 integrin and desmin have been used as markers for these earlier stages of myogenesis. Both proteins are expressed in replicating secondary myoblasts prior to terminal differentiation and when these cells differentiate further, the expression of the alpha 7 integrin and desmin genes is up-regulated. To determine whether the stages of myogenesis which precede terminal development and the factors which regulate them are distinct, the expression of alpha 7 integrin and desmin was assayed in a variety of myogenic cell lines in which terminal differentiation was inhibited. L8E63 and C2 myoblasts in which terminal differentiation was inhibited by SV40 large T antigen, adenovirus E1A protein, or ras and an L6 mutant whose terminal differentiation is sensitive to alpha-amanitin were studied. In all cases, when terminal myogenic differentiation is inhibited the basal levels of desmin and alpha 7 expression are not altered. Under these same conditions expression of the myogenic regulatory genes myogenin and MyoD also were inhibited whereas Myf-5 persisted. These results indicate that expression of the early myogenic phenotype and terminal differentiation are discrete and independent stages of myogenesis and that different transcription factors likely regulate the expression of each stage. In contrast with myoblasts in cultures of newborn rat hindlimb cells and the C2 cell line, myogenic cells derived from C3H10T1/2 cells by treatment with 5-azacytidine or by transfection with MyoD, Myf-5, or MRF4 do not express desmin as replicating myoblasts but do so upon terminal differentiation. This indicates that in vitro, terminal differentiation can proceed in the absence of the phenotypes that normally develop earlier and that the conversion of 10T1/2 cells to myogenic cells can bypass developmental stages which normally occur in vivo. These results are discussed in the context of a model of the myogenic lineage that is based on the expression of desmin.