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PubMed2003

Downstream coding region determinants of bacterio-opsin, muscarinic acetylcholine receptor and adrenergic receptor expression in Halobacterium salinarum.

The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum.

Amino Acid Sequence↗
PubMed2000

Export of mercury downstream from reservoirs.

Environmental effects monitoring at the La Grande hydroelectric complex (Québec, Canada) revealed important increases in mercury levels in fish caught downstream from reservoirs. A study was carried out in 1997 immediately below the Caniapiscau Reservoir to identify by which components mercury is transported downstream from reservoirs and to assess the amount of mercury being exported. Stomach contents of lake whitefish (Coregonus clupeaformis) captured immediately below the Caniapiscau Reservoir were examined to determine which components transfer mercury from lower trophic levels to fish. The analyses of water samples and drifting organisms collected below the reservoir indicate that the dissolved fraction (< 0.45 microm) and the suspended particulate matter (0.45-50 microm) are the major components by which methylmercury is transferred downstream of reservoirs, accounting for 64 and 33%, respectively, of the total amounts exported. Drifting organisms such as plant debris, benthic invertebrates, fish, phytoplankton and zooplankton are much less important pathways for mercury export because of their very low biomass per water volume coming out of the generating station, as opposed to the high biomass of suspended particulate matter. However, zooplankton is the major component by which methylmercury is directly transferred to non-piscivorous fish downstream.

Animals↗
PubMed2000

Kluyveromyces marxianus exhibits an ancestral Saccharomyces cerevisiae genome organization downstream of ADH2.

In Saccharomyces cerevisiae, the alcohol dehydrogenase genes ADH1 and ADH5 are part of a duplicated block of genome, thought to originate from a genome-wide duplication posterior to the divergence from the Kluyveromyces lineage. We report here the characterization of Kluyveromyces marxianus ADH2 and the five genes found in its immediate downstream region, MRPS9, YOL087C, RPB5, RIB7 and SPP381. The order of these six genes reflects the structure of the ancestral S. cerevisiae genome before the duplication that formed the blocks including ADH1 on chromosome XV and ADH5 on chromosome II, indicating these ADH genes share a direct ancestor. On the one hand, the two genes found immediately downstream of KmADH2 are located, for the first, downstream ADH5 and, for the second, downstream ADH1 in S. cerevisiae. On the other hand, the order of the paralogs included in the blocks of ADH1 and ADH5 in S. cerevisiae suggests that two of them have been inverted within one block after its formation, and that inversion is confirmed by the gene order observed in K. marxianus.

Alcohol Dehydrogenase↗
PubMed1998

The low level expression of chloramphenicol acetyltransferase (CAT) mRNA in Escherichia coli is not dependent on either Shine-Dalgarno or the downstream boxes in the CAT gene.

Recent studies have shown that the canonical Shine-Dalgarno (SD)-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. Alternative non-SD sequences (located upstream from the initiation codon) and also downstream sequences ("downstream boxes") complementary to 16S rRNA were found to be involved in the initiation of translation of mRNAs devoid of either SD or any leader sequences. In this study the chloramphenicol acetyltransferase (CAT) gene was modified to remove the 5' terminal non-translated region and/or the two potential downstream boxes in the CAT gene. Thus a series of ten CAT gene constructs was created and expressed in E. coli under a strong constitutive promoter. The results showed that CAT mRNAs devoid of both leader sequence nucleotides and the two downstream boxes in the CAT gene remained active in vivo and produced CAT protein in sufficient amounts for survival of the transformed cells at chloramphenicol concentrations up to 20-30 micrograms/ml.

5' Untranslated Regions↗
PubMed2001

The involvement of sex steroid hormones in downstream and upstream migratory behavior of masu salmon.

From May through July when masu salmon, Oncorhynchus masou, commence downstream migration under natural conditions, yearling precocious male masu salmon (resident form) showed higher GSI and plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) in contrast to immature smolts (migratory form). From March through September coinciding with the upstream migration period, 2-year-old male and female adults also showed higher GSI and plasma levels of T, estradiol-17beta (E(2)) 11-KT, 17alpha-hydroxyprogesterone and 17alpha,20beta-dihydroxy-4-pregnene-3-one (DHP). In order to test the effects of steroid hormones on migratory behaviors, silascone tube capsules containing 500 microg of T, E(2), 11-KT, DHP, or a vehicle was implanted into smolts, castrated precocious males, or immature parr, and downstream and upstream behavior were observed in artificial raceways in spring and autumn. Downstream behavior of smolts was inhibited significantly by T, E(2) and 11-KT. Upstream behavior was stimulated by T and 11-KT in castrated precocious males and stimulated by T, E(2) and 11-KT in immature parr. These results indicate that T, E(2) and 11-KT are the factors regulating downstream and upstream migratory behavior. In particular, because of its changing patterns in plasma and significant effects, T, the common precursor hormone of E(2) (female) and 11-KT (male), is considered to play central roles in both types of behavior.

Animals↗
PubMed2002

Identification of an essential pseudoknot in the putative downstream internal ribosome entry site in giardiavirus transcript.

Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (DB) complementary to the anti-DB at the 3' end of 16S-like rRNA, (2) stem-loops I, II, III, and IVA, and (3) a pentanucleotide 5'-UCUCC-3' immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5'-GGAGA-3' in loop II. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson-Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1-6 nt) 5' untranslated regions in its mRNAs.

Animals↗
PubMed2006

Diversity of epidermal growth factor receptor-mediated activation of downstream molecules in human lung carcinomas.

The correlations among epidermal growth factor receptor (EGFR) gene amplification, gene mutation, overexpression/phosphorylation of EGFR protein and activation of its downstream molecules, signal transducers and activators of transcription 3 (Stat-3), Akt and extracellular signal-related protein kinase 1/2 (Erk1/2) were investigated in 28 cases of human lung carcinomas. In five cases of carcinomas with EGFR amplification, EGFR expression and phosphorylation levels were higher than other cases, and Stat-3 was activated in all five cases. Point mutations in the tyrosine kinase domain of EGFR were detected in five cases, one of which was also associated with gene amplification. In these five cases, both EGFR expression and phosphorylation were enhanced, and Akt was activated in four cases. In the remaining 19 cases, EGFR protein expression was upregulated in eight cases and phosphorylated in four cases, but neither EGFR nor phosphorylated-EGFR expression levels specifically correlated with activation of particular downstream molecules. In general, either Stat-3 or Akt, but not both, was activated reciprocally and complementarily to each other, as indicated by their phosphorylation. However, Erk1/2 was activated regardless of the status of Stat-3, Akt or EGFR proteins. The current data suggest that persistent Stat-3 activation may be a critical event downstream of EGFR that has been overexpressed by gene amplification. In contrast, tumor cells harboring the EGFR mutation may persistently activate a cascade via Akt. Finally, in the majority of cases that have no aberration of the EGFR, its downstream molecules function in reciprocal and/or complementary manner in the maintenance and/or progression of carcinomas. These overall results could provide novel insights into potential chemotherapeutic regimens for lung carcinomas, such as inhibitors of Stat-3, Akt and Erk1/2.

Carcinoma, Non-Small-Cell Lung↗
PubMed1996

Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor.

BACKGROUND: Oct-3, a pluripotent cell-specific POU transcription factor, appears to be a key regulator in pluripotential early embryonic cells and germ cells. In order to study how pluripotency is maintained, it is essential to know what genes are regulated by Oct-3. RESULTS: By employing a subtraction method, we identified several pluripotent cell-specific genes. Based upon expression patterns in various cell lines lacking or possessing Oct-3 function, about half of the genes were placed downstream of Oct-3. These downstream genes included a previously-known gene (Glut-3: a gene for a glucose transporter) and novel genes (226, 383 and 880). Their expression patterns paralleled that of Oct-3: all of these genes were highly expressed in pluripotent cells such as EC/ES cells, but switched off upon differentiation. More importantly, their expression was rescued in 'revertant' cells that ectopically acquired the Oct-3 transactivating function. Furthermore, the expression profiles of Glut-3, 226 and 383 during mouse development also overlapped that of Oct-3. The Glut-3 gene possessed multiple Oct-3 binding sites in its transcriptional regulatory regions, suggesting that at least one of the downstream genes was a direct target of Oct-3. CONCLUSIONS: A large proportion of pluripotent cell-specific genes appear to be downstream targets of Oct-3.

Amino Acid Sequence↗
PubMed1993

GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.

Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells.

3T3 Cells↗
PubMed1996

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.

Azides↗
PubMed1995

A conserved downstream element defines a new class of RNA polymerase II promoters.

Although many TATA-less promoters transcribed by RNA polymerase II initiate transcription at multiple sites, the regulation of multiple start site utilization is not understood. Beginning with the prediction that multiple start site promoters may share regulatory features and using the P-glycoprotein promoter (which can utilize either a single or multiple transcription start site(s)) as a model, several promoters with analogous transcription windows were grouped and searched for the presence of a common DNA element. A downstream protein-binding sequence, MED-1 (Multiple start site Element Downstream), was found in the majority of promoters analyzed. Mutation of this element within the P-glycoprotein promoter reduced transcription by selectively decreasing utilization of downstream start sites. We propose that a new class of RNA polymerase II promoters, those that can utilize a distinctive window of multiple start sites, is defined by the presence of a downstream MED-1 element.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Protein kinase C activates the plasma membrane Ca2+ pump isoform 4b by phosphorylation of an inhibitory region downstream of the calmodulin-binding domain.

The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.

Amino Acid Sequence↗
PubMed2003

Tumor-related alternatively spliced Rac1b is not regulated by Rho-GDP dissociation inhibitors and exhibits selective downstream signaling.

Rac1 is a member of the Rho family of small GTPases, which control signaling pathways that regulate actin cytoskeletal dynamics and gene transcription. Rac1 is activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. In addition, Rho-GDP dissociation inhibitors (Rho-GDIs) can inhibit Rac1 by sequestering it in the cytoplasm. We have found previously that colorectal tumors express an alternatively spliced variant, Rac1b, containing 19 additional amino acids following the switch II region. Here we characterized the regulation and downstream signaling of Rac1b. Although little Rac1b protein is expressed in cells, the amount of activated Rac1b protein often exceeds that of activated Rac1, suggesting that Rac1b contributes significantly to the downstream signaling of Rac in cells. The regulation of both Rac1 and Rac1b activities is dependent on guanine nucleotide exchange factors and GTPase-activating proteins, but the difference in their activation is mainly determined by the inability of Rac1b to interact with Rho-GDI. As a consequence, most Rac1b remains bound to the plasma membrane and is not sequestered by Rho-GDI in the cytoplasm. Unlike Rac1, activated Rac1b is unable to induce lamellipodia formation and is unable to bind and activate p21-activated protein kinase nor activate the downstream protein kinase JNK. However, both Rac1 and Rac1b are able to activate NFkappaB to the same extent. These data suggest that alternative splicing of Rac1 leads to a highly active Rac variant that differs in regulation and downstream signaling.

Alternative Splicing↗
PubMed2005

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling.

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.

Animals↗
PubMed2005

Receptor activator of NF-kappaB (RANK) cytoplasmic motif, 369PFQEP373, plays a predominant role in osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4.

Receptor activator of NF-kappaB ligand (RANKL) plays a crucial role in osteoclast differentiation, function, and survival. RANKL exerts its effect by activating its receptor RANK (receptor activator of NF-kappaB), which recruits various intracellular signaling molecules via specific motifs in its cytoplasmic tail. Previously, we identified three RANK cytoplasmic motifs (Motif 1, 369PFQEP373; Motif 2, 559PVQEET564; and Motif 3, 604PVQEQG609) mediating osteoclast formation and function. Here, we investigated RANK cytoplasmic motifs involved in osteoclast survival. Motif 1, in contrast to its minimal role in osteoclast formation and function, plays a predominant role in promoting osteoclast survival. Moreover, whereas Motif 2 and Motif 3 are highly potent in osteoclast formation and function, they exert a moderate effect on osteoclast survival. We also investigated the role of these motifs in activating Akt/protein kinase B (PKB), which has been implicated in RANKL-induced osteoclast survival. Motif 1, but not Motif 2 or Motif 3, is able to stimulate Akt/PKB activation. Because Akt/PKB has been shown to utilize distinct downstream effectors (glycogen synthase kinase-3beta, FKHR/FOXO1a, BAD, and AFX/FOXO4) to regulate cell survival, we next determined which downstream effector(s) is activated by Akt/PKB to promote osteoclast survival. Our data revealed that RANKL only stimulates AFX/FOXO4 phosphorylation, indicating that AFX/FOXO4 is a key downstream target activated by Akt/PKB to modulate osteoclast survival. Taken together, we conclude that Motif 1 plays a predominant role in mediating osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4.

Amino Acid Motifs↗
PubMedDate not supplied

Comparison of pitot traverses taken at varying distances downstream of obstructions.

This study determined the deviations between pitot traverses taken under "ideal" conditions--at least seven duct diameter's lengths (i.e., distance = 7D) from obstructions, elbows, junction fittings, and other disturbances to flows--with those taken downstream from commonplace disturbances. Two perpendicular 10-point, log-linear velocity pressure traverses were taken at various distances downstream of tested upstream conditions. Upstream conditions included a plain duct opening, a junction fitting, a single 90 degrees elbow, and two elbows rotated 90 degrees from each other into two orthogonal planes. Airflows determined from those values were compared with the values measured more than 40D downstream of the same obstructions under ideal conditions. The ideal measurements were taken on three traverse diameters in the same plane separated by 120 degrees in honed drawn-over-mandrel tubing. In all cases the pitot tubes were held in place by devices that effectively eliminated alignment errors and insertion depth errors. Duct velocities ranged from 1500 to 4500 ft/min. Results were surprisingly good if one employed two perpendicular traverses. When the averages of two perpendicular traverses was taken, deviations from ideal value were 6% or less even for traverses taken as close as 2D distance from the upstream disturbances. At 3D distance, deviations seldom exceeded 5%. With single diameter traverses, errors seldom exceeded 5% at 6D or more downstream from the disturbance. Interestingly, percentage deviations were about the same at high and low velocities. This study demonstrated that two perpendicular pitot traverses can be taken as close as 3D from these disturbances with acceptable (< or = 5%) deviations from measurements taken under ideal conditions.

Air Movements↗
PubMed2002

Groundwater contamination downstream of a contaminant penetration site. II. Horizontal penetration of the contaminant plume.

Part I of this study (Rubin, H.; Buddemeier, R.W. Groundwater Contamination Downstream of a Contaminant Penetration Site Part 1: Extension-Expansion of the Contaminant Plume. J. of Environmental Science and Health Part A (in press).) addressed cases, in which a comparatively thin contaminated region represented by boundary layers (BLs) developed within the freshwater aquifer close to contaminant penetration site. However, at some distance downstream from the penetration site, the top of the contaminant plume reaches the top or bottom of the aquifer. This is the location of the "attachment point," which comprises the entrance cross section of the domain evaluated by the present part of the study. It is shown that downstream from the entrance cross section, a set of two BLs develop in the aquifer, termed inner and outer BLs. It is assumed that the evaluated domain, in which the contaminant distribution gradually becomes uniform, can be divided into two sections, designated: (a) the restructuring section, and (b) the establishment section. In the restructuring section, the vertical concentration gradient leads to expansion of the inner BL at the expense of the outer BL, and there is almost no transfer of contaminant mass between the two layers. In the establishment section, each of the BLs occupies half of the aquifer thickness, and the vertical concentration gradient leads to transfer of contaminant mass from the inner to the outer BL. By use of BL approximations, changes of salinity distribution in the aquifer are calculated and evaluated. The establishment section ends at the uniformity point, downstream from which the contaminant concentration profile is practically uniform. The length of the restructuring section, as well as that of the establishment section, is approximately proportional to the aquifer thickness squared, and is inversely proportional to the transverse dispersivity. The study provides a convenient set of definitions and terminology that are helpful in visualizing the gradual development of uniform contaminant concentration distribution in an aquifer subject to contaminant plume penetration. The method developed in this study can be applied to a variety of problems associated with groundwater quality, such as initial evaluation of field data, design of field data collection, the identification of appropriate boundary conditions for numerical models, selection of appropriate numerical modeling approaches, interpretation and evaluation of field monitoring results, etc.

Environmental Monitoring↗
PubMed1997

Enhancer complexes located downstream of both human immunoglobulin Calpha genes.

To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Calpha genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Calpha1 and Calpha2 genes we identified several segments of DNA which display B lymphoid-specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Calpha genes are nearly identical to each other. These enhancers are also homologous to three regions which lie in similar positions downstream from the murine Calpha gene and form the murine LCR. The strongest enhancers in both mouse and human have been designated HS12. Within a 135-bp core homology region, the human HS12 enhancers are approximately 90% identical to the murine homolog and include several motifs previously demonstrated to be important for function of the murine enhancer; additional segments of high sequence conservation suggest the possibility of previously unrecognized functional motifs. On the other hand, certain functional elements in the murine enhancer, including a B cell-specific activator protein site, do not appear to be conserved in human HS12. The human homologs of the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but for at least two of the functional motifs in the murine HS4 (a kappaB site and an octamer motif ) the human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes a region of high sequence conservation with mouse, suggesting the possibility of another novel functional element.

Amino Acid Sequence↗