Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout.

A rainbow trout major heat-shock-protein-like gene (hsp 70) and corresponding cDNA clones were isolated by hybridization to heterologous hsp70 probes. DNA sequencing revealed that this gene is structurally similar to a mammalian heat-shock-cognate hsc70 gene and consists of eight introns. Northern blot and primer extension analyses showed that the corresponding mRNA is constitutively abundant in different trout tissues and salmonid cell lines. Fragments of the isolated gene containing the -900 - +30 and -217 - +58 sequence were linked to a bacterial chloramphenicol acetyltransferase reporter gene and transiently transfected into salmonid cells. The expression pattern of these constructs supports our conclusion that the isolated genomic and cDNA clones correspond to a trout heat-shock-cognate hsc70 gene.     

Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.     

Firefly luciferase gene: structure and expression in mammalian cells.

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Regulation of neurotransmitter synthesis: from neuron to gene.

We are interested in the molecular mechanisms of the regulation of neurotransmitter related gene expression by neurotrophic factors and neuronal activity. Previous work has shown that conditioned medium of muscle cells induces choline acetyltransferase (CAT) activity and represses tyrosine hydroxylase, dopamine-beta-hydroxylase and aromatic L-amino acid decarboxylase (AADC) activities in cultured sympathetic neurons. Here, we show that a new muscle-derived cell line secretes two factors which induce CAT activity in spinal cord cultures; one of those is related to LIF, a CAT inducing factor for sympathetic neurons. Preliminary data are presented on the structure of the human AADC and CAT genes. Putative promoter regions have been coupled to reporter genes; transient transfection experiments will allow us to determine the promoter elements responsible for the regulation by neurotrophic factors. We also summarize the distribution of AADC-immunoreactive cells in rat and cat brain, which will be used as a reference for the study of the specificity of expression of AADC promoter in transgenic mice.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

A mouse homeobox containing gene on chromosome 11: sequence and tissue-specific expression.

We have molecularly cloned a mouse homeobox containing gene by isolating cDNA and genomic clones. The gene is located in a previously described cluster on chromosome 11 (Hart et al. (1985) Cell 43, 9-18) and was identified as the Hox2.3 gene. We present the complete mRNA sequence of this gene and describe similarities to other homeobox containing genes, among which its human homologue, the cl gene. High expression of the Hox2.3 gene was found in kidney, testis, and spinal cord of adult mice, in the spinal cord of 12.5-17.5 day embryos and in differentiating EC cells depending on their treatment. Three different treatments of the pluripotent EC cell line P19, each leading to the induction of a specific differentiation pathway, resulted in all cases in induction of Hox2.3; however, major quantitative differences in this response were observed.     

Sequence and expression of a novel mouse gene PRDC (protein related to DAN and cerberus) identified by a gene trap approach

Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ-line transmitted and X-gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening-selected gene aberrative in neuroblastoma) and cerberus . (DAN is a possible tumor-suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.