Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C alpha 1 and C alpha 2 genes

We have isolated sequences downstream of human Ig C alpha 1 and C alpha 2 genes and have identified two enhancers in these regions. One enhancer is located approximately 9 kb downstream of C alpha 1, and the second enhancer is located approximately 11 kb downstream of C alpha 2. These approximately 1.6-kb enhancers are virtually identical to each other except for varying numbers of a approximately 53-bp motif. The C alpha 2-associated enhancer contains four copies of this motif in tandem, whereas the C alpha 1-associated enhancer has only a single copy. Within the human enhancers is a 177-bp segment that is homologous to a 191-bp segment of one of four enhancers from the 3' regulatory region of murine (and rat) DNA, namely 3'IgH-E(hs1,2). Like the murine and rat enhancers, both human enhancers are flanked by inverted repeats; furthermore, the human enhancers generally appear to be inverted with respect to each other. The evolutionarily conserved region of homology has substantial core enhancer activity. Contained within this region are the single octamer and one copy of the approximately 53-bp motif, both of which contribute to the activity of the full-length enhancer. A comparison of the DNA sequences and the results of transient transfection assays imply that the human C alpha-associated enhancers may be regulated (in part) differently than the murine enhancer 3'IgH-E(hs1,2).     

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.     

The histostructure of the adrenal gland in the Campbell hamster after extirpation of a specific midventral gland]

The receptors for retinoic acid (RA) and for 1 alpha,25-dihydroxyvitamin D3 (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-alpha type 1 receptor (TNF alpha RI) gene identified a 3' enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein-protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD.     

Lipase specificity toward some acetylenic and olefinic alcohols in the esterification of pentanoic and stearic acids

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trip1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74.1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region.     

Upstream and downstream cis-acting elements for cleavage at the L4 polyadenylation site of adenovirus-2

A study of the cis-acting elements involved in the 3' end formation of the RNAs from the major late L4 family of adenovirus-2 was undertaken. Series of 5' or 3' end deletion mutants and mutants harboring either internal deletions or substitutions were prepared and assayed for in vitro cleavage. This first allowed the demonstration of a sequence, located at -6 to -29, relative to AAUAAA, whose deletion or substitution reduces cleavage efficiency at the L4 polyadenylation site two to three fold. This upstream efficiency element 5' AUCUUUGUUGUC/AUCUCUGUGCUG 3' is constituted of a partially repeated 12 nucleotide long, UCG rich sequence. The activities of the 2 sequence elements in cleavage are additive. We also searched for regulatory sequences downstream of the L4 polyadenylation site. We found that the deletion or substitution of a 30 nucleotide long UCG rich sequence, between nucleotides +7 and +35 relative to the cleavage site and harboring a UCCUGU repeat reduces cleavage efficiency at least ten fold. A GUUUUU sequence, starting at +35 had no influence. Thus, the usage of the L4 polyadenylation site requires down-stream sequences different from the canonical GU or U boxes and is regulated by upstream sequence elements.     

Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.