Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

Golgi alpha-mannosidase II (alpha-MII) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human alpha-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding alpha-MII were isolated. One such gene was found to encode an isozyme, designated alpha-MIIx. A 5-kb cDNA clone encoding alpha-MIIx was then isolated from a human melanoma cDNA library. However, comparison between alpha-MIIx and alpha-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while alpha-MII consists of 1144 amino acid residues. To reevaluate the sequence of alpha-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the alpha-MIIx genomic sequence revealed that alternative splicing of the alpha-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of alpha-MIIx in various tissues, suggesting that the alpha-MIIx gene is a housekeeping gene. COS cells transfected with alpha-MIIx cDNA containing the full-length open reading frame showed an increase of alpha-mannosidase activity. The alpha-MIIx gene was mapped to human chromosome 15q25, whereas the alpha-MII gene was mapped to 5q21-22.

Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity.

The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the Sp1 binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far-upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor Sp1.

Analysis of perinatal gene expression: hormone response elements mediate activation of a lacZ reporter gene in liver of transgenic mice.

The transcription of genes encoding gluconeogenic enzymes is tightly regulated during the perinatal period. These genes are induced by glucagon (cAMP) and glucocorticoids and repressed by insulin. To address the role of cAMP and glucocorticoids in the physiological activation of genes encoding gluconeogenic enzymes in the perinatal period, transgenic mice have been generated with chimeric constructs containing the reporter gene lacZ under the control of hormone response elements. The activity of the transgene is restricted to the liver by the presence of the enhancers from the alpha-fetoprotein gene and its transcription is driven by a promoter that contains a TATA box linked to either cAMP response elements (CREs) or glucocorticoid response elements (GREs). We demonstrate cAMP and glucocorticoid regulation, liver-specific expression, and perinatal activation of the reporter gene. These data indicate that the CRE and GRE are, independently, necessary and sufficient to mediate perinatal gene activation. Perinatal activation was not impaired when a CRE reporter transgene was assayed in mice that contain a targeted mutation of the CRE-binding protein (CREB) gene, providing further evidence for functional redundancy among the members of the CREB/ATF gene family.

Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth.

To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae. The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids). Disruption of the yeast alpha-COP gene is lethal. Comparison of the DNA-derived primary structure with peptides from bovine alpha-COP shows a striking homology. alpha-COP is localized to coated transport vesicles and coated buds of Golgi membranes derived from CHO cells.

The herpes simplex virus major regulatory protein ICP4 blocks apoptosis induced by the virus or by hyperthermia.

Cells infected with herpes simplex virus 1 (HSV-1) undergo productive or latent infection without exhibiting features characteristic of apoptosis. In this report, we show that HSV-1 induces apoptosis but has evolved a function that blocks apoptosis induced by infection as well as by other means. Specifically, (i) Vero cells infected with a HSV-1 mutant deleted in the regulatory gene alpha 4 (that encodes repressor and transactivating functions), but not those infected with wild-type HSV-1(F), exhibit cytoplasmic blebbing, chromatin condensation, and fragmented DNA detected as a ladder in agarose gels or by labeling free DNA ends with terminal transferase; (ii) Vero cells infected with wild-type HSV-1(F) or cells expressing the alpha 4 gene and infected with the alpha 4- virus did not exhibit apoptosis; (iii) fragmentation of cellular DNA was observed in Vero cells that were mock-infected or infected with the alpha 4- virus and maintained at 39.5 degrees C, but not in cells infected with wild-type virus and maintained at the same temperature. Wild-type strains of HSV-1 with limited extrahuman passages, such as HSV-1 (F), carry a temperature-sensitive lesion in the alpha 4 gene and at 39.5 degrees C only alpha genes are expressed. These results indicate that the product of the alpha 4 gene is able to suppress apoptosis induced by the virus as well by other means.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene.

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.