L-arginine increases plasma homocysteine in apoE-/-/iNOS-/- double knockout mice.

Previous studies have shown that L-arginine (L-Arg) administration to apoE-/-/iNOS-/- double knockout mice (dKO) on a Western diet paradoxically results in an increase in atherosclerotic lesion size. We hypothesized that the potential beneficial effects of L-Arg could be offset, in part, by the byproducts of L-Arg catabolism, especially the atherogenic risk factor, homocysteine. In the kidney, L-Arg is converted to L-ornithine and guanidinoacetate (GAA) by L-arginine-glycine amidinotransferase. The efficient transmethylation of GAA by an S-adenosyl-methionine (SAM)-dependent methyltransferase in liver yields creatine and S-adenosylhomocysteine (SAH), which is readily hydrolyzed to homocysteine and adenosine. We, therefore, measured total plasma homocysteine in the dKO mice and control mice. We found that L-Arg supplementation caused a 37% increase in total plasma homocysteine (tHcy) levels in dKO mice compared to controls not treated with L-Arg (5.2+/-2.2 vs 3.8+/-1.5 microM Hcy, p<0.04). In a liver cell line, HepG2, addition of 10 and 50 microM GAA in the presence of 50 microM L-methionine (L-Met) increased tHcy production by approximately 1.47 (p<0.0001) and 2.3-fold (p<0.0001), respectively. In the presence of additional 100 microM L-Met, baseline homocysteine production was elevated by 20% (p<0.005), and 10 and 50 microM GAA augmented homocysteine production by an additional 1.88- (p<0.0001) and 3.4-fold (p<0.001), respectively, compared with 50 microM L-Met. These data suggest that increased concentrations of a methyl acceptor, such as L-Arg-derived GAA, drives SAM-dependent-methylation and consequent homocysteine formation. Furthermore, L-Met levels can also influence homocysteine production likely by regulating the synthesis of the methyl donor SAM. Epidemiological studies have suggested that homocysteine is a graded risk factor. In animal models, modestelevations of homocysteine can cause endothelial dysfunction and augment atherosclerosis. Our data suggest that L-arginine supplementation may contribute to vascular injury and atherogenesis under some circumstances by elevating homocysteine levels.

Role of the postsynaptic alpha(2)-adrenergic receptor subtypes in catecholamine-induced vasoconstriction.

Catecholamines induce direct vasoconstriction mediated by postsynaptic alpha-adrenergic receptors (alpha-ARs) of both the alpha(1) and alpha(2) type. To evaluate the contribution of each alpha(2)-AR subtype (alpha(2A), alpha(2B), and alpha(2C)) to this function, we used groups of genetically engineered mice deficient for the gene to each one of these subtypes and compared their blood pressure (BP) responses to their wild-type counterparts. Blood pressure responses to a bolus of norepinephrine (NE) were assessed before and after sequential blockade of alpha(1)-ARs with prazosin and alpha(2)-ARs with yohimbine. The first NE bolus elicited a brief 32 to 44 mm Hg BP rise (p < 0.001 from baseline) in all six groups. Prazosin decreased BP by 23 to 33 mm Hg in all groups, establishing a new lower baseline. Repeat NE at that point elicited lesser but still significant (p < 0.001) brief pressor responses between 32% and 45% of the previous BP rise in five of the six groups. Only the alpha(2A)-AR gene knockouts differed, responding instead with a 20-mm Hg fall in BP, a significant change from baseline (p < 0.001) and different from the pressor response of their wild-type counterparts (p < 0.001). The addition of yohimbine produced no further BP change in the five groups, but it did produce a small 7. 5-mm Hg fall (p < 0.05) in the alpha(2A)-AR knockouts. Norepinephrine bolus during concurrent alpha(1) and alpha(2)-AR blockade produced significant (p < 0.001) hypotensive responses in all subgroups, presumably attributable to unopposed stimulation of beta(2)-vascular wall ARs. We conclude that the alpha(2)-AR-mediated vasoconstriction induced by catecholamines is attributable to the alpha(2A)-AR subtype because mice deficient in any one of the other subtypes retained the capacity for normal vasoconstrictive responses. However, the alpha(1)-ARs account for the major part (as much as 68%) of catecholamine-induced vasoconstriction.

Identification of a polymorphic glutamic acid stretch in the alpha2B-adrenergic receptor and lack of linkage with essential hypertension.

Essential hypertension, a clinically significant elevation in blood pressure with no recognizable cause, is believed to be attributable to the collective effect of genetic predisposing factors in combination with specific environmental factors, such as diet and stress. Of the genetic causes, genes coding for proteins involved in blood pressure regulation, such as the alpha- and beta-adrenergic receptors, are obvious candidates. The alpha2-adrenergic receptor plays a key role in the sympathetic nervous system by mediating the effects of epinephrine and norepinephrine. To evaluate the potential role between the alpha2B receptor and essential hypertension, we scanned the alpha2B-receptor gene for genetic variation in 108 affected sibling pairs. The screening revealed two major forms of the receptor. They differ by the presence of either 9 or 12 glutamic acid residues in the acidic domain of the third cytoplasmic loop of the protein. Investigation of the pattern of this variation in hypertensive sibling pairs suggests that the alpha2B receptor locus does not contribute substantially to genetic susceptibility for essential hypertension.

Sympathoinhibitory function of the alpha(2A)-adrenergic receptor subtype.

Presynaptic alpha(2)-adrenergic receptors (alpha(2)-AR) are distributed throughout the central nervous system and are highly concentrated in the brain stem, where they contribute to neural baroreflex control of blood pressure (BP). To explore the role of the alpha(2A)-AR subtype in this function, we compared BP and plasma norepinephrine and epinephrine levels in genetically engineered mice with deleted alpha(2A)-AR gene to their wild-type controls. At baseline, the alpha(2A)-AR gene knockouts (n=11) versus controls (n=10) had higher systolic BP (123+/-2.5 versus 115+/-2.5 mm Hg, P<0. 05), heart rate (730+/-15 versus 600+/-18 b/min, P<0.001), and norepinephrine (1.005+/-0.078 versus 0.587+/-0.095 ng/mL, P<0.01), respectively. When submitted to subtotal nephrectomy and given 1% saline as drinking water, both alpha(2A)-AR gene knockouts (n=14) and controls (n=14) became hypertensive, but the former required 15. 6+/-2.5 days versus 29.3+/-1.4 days for the controls (P<0.001). End-point systolic BP was similar for both at 155+/-2.1 versus 152+/-5.2 mm Hg, but norepinephrine and epinephrine levels were twice as high in the knockouts at 1.386+/-0.283 and 0.577+/-0.143 versus 0.712+/-0.110 and 0.255+/-0.032 ng/mL, respectively, P<0.05 for both. We conclude that the alpha(2A)-AR subtype exerts a sympathoinhibitory effect, and its loss leads to a hypertensive, hyperadrenergic state.

Role of the alpha2B-adrenergic receptor in the development of salt-induced hypertension.

Salt sensitivity is a common trait in patients with essential hypertension and seems to have both an inherited and an acquired component (eg, is influenced by aging and renal insufficiency). Experimental evidence suggests that salt loading induces hypertension via a neurogenic mechanism mediated by the alpha2-adrenergic receptors (alpha2-AR). To explore the alpha2-AR subtype involved in this mechanism, we studied 2 groups of mice genetically engineered to be deficient in one of the 3 alpha2-AR subtype genes (either alpha2B-AR +/- or alpha2C-AR -/- knockout mice) compared with their wild-type counterparts. The mice (n=10 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for up to 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intra-arterial BP recording. The alpha2B-AR-deficient mice had an attenuated BP response in this protocol (baseline 101.8+/-2.7 versus end point 109.9+/-2.8 mm Hg), whereas the BP of their wild-type counterparts went from a baseline 101.9+/-2.3 to an end point 141.4+/-7.1 mm Hg. The other 2 groups had BP increases of 44. 6+/-5.17 and 46.7+/-7.01 mm Hg, with no difference between the mice deficient in the alpha2C-AR gene subtype versus their wild-type counterparts. Body weight, renal remnant weight, and residual renal function were no different among groups. These data suggest that a full complement of alpha2B-AR genes is necessary to raise BP in response to dietary salt loading, whereas complete absence of the alpha2C-AR subtype does not preclude salt-induced BP elevation. It is unclear whether the mechanism(s) involved in this process are of central origin (inability to increase sympathetic outflow), vascular origin (inability to vasoconstrict), or renal origin (inability to retain excess salt and fluid).

Autosomal dominant orthostatic hypotensive disorder maps to chromosome 18q.

Familial orthostatic hypotensive disorder is characterized by light-headedness on standing, which may worsen to syncope, palpitations, and blue-purple ankle discoloration, and is accompanied by a marked decrease in systolic blood pressure, an increase in diastolic pressure, and tachycardia, all of which resolve when supine. We ascertained three families in which this disorder is inherited as an autosomal dominant trait with reduced penetrance. A genomewide scan was conducted in the two largest families, and three regions with multipoint LOD scores >1.5 were identified. Follow-up of these regions with additional markers in all three families yielded significant evidence of linkage at chromosome 18q. A maximum multipoint LOD score of 3.21 in the three families was observed at D18S1367, although the smallest family had negative LOD scores in the entire region. There was significant evidence of linkage in the presence of heterogeneity at 18q, with a maximum LOD score of 3.92 at D18S1367 in the two linked families. Identification of the gene responsible for orthostatic hypotensive disorder in these families may advance understanding of the general regulatory pathways involved in the continuum, from hypotension to hypertension, of blood pressure.

Expression of alpha2-adrenergic receptors in normal and atherosclerotic rabbit aorta.

Alpha2-adrenergic receptors (alpha2-ARs) in vascular smooth muscle cells are known to mediate vasoconstriction; however, it is unknown which of the 3 subtypes of alpha2-AR (alpha2A, alpha2B, or alpha2C) is expressed in vascular tissue. We have used subtype-specific probes in in situ hybridization and RNase protection assays to analyze the expression of alpha2-AR in the thoracic aorta of New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits, a model for atherosclerosis. We found that the alpha2A-AR mRNA was in endothelial and smooth muscle cells in both NZW and WHHL aorta. In addition, the shoulders and subendothelial regions of the atherosclerotic lesions in WHHL aorta showed abundant expression of alpha2A-AR mRNA. Antibodies to macrophage (RAM-11) and smooth muscle cell (HHF-35) antigens were used to localize macrophage and smooth muscle cells in aortic sections from WHHL rabbits. The expression of alpha2A-AR mRNA within the lesions of WHHL rabbits correlated with the presence of infiltrating macrophages. We discuss the potential role of alpha2A-ARs in macrophage function and in promoting atherosclerosis.

Models of experimental hypertension in mice.

Experimental models of hypertension in various animals are useful in the research of vasoactive mechanisms. Recombinant DNA technology has produced genetically engineered animals, mostly mice, useful in hypertension research. However, the development of hypertensive models in mice is fraught with technical difficulties. We describe here the successful development in mice of two common types of experimental hypertension: the renovascular two-kidney, one clip and mineralocorticoid deoxycorticosterone-salt models. By adapting technology previously used in rats, we succeeded in developing hypertension (defined as systolic pressures higher than 140 mm Hg) in more than 50% of mice so treated. We also adapted the methodology for indirect tail-cuff blood pressure measurements as well as for direct intra-arterial monitoring of blood pressure in conscious, freely moving mice. Application of these techniques in transgenic or gene knockout mice with altered vasoactive hormones or receptors should allow elucidation of the role of the target gene products in various types of hypertension.

Evidence for cell-specific regulation of transcription of the rat alpha2A-adrenergic receptor gene.

We investigated the transcriptional activity of the -131 to -92 region of the rat alpha2A-adrenergic receptor gene. In HT29 cells, this region has a positive effect on transcription, whereas in RINm5F cells, this region has a negative effect on transcription. The -131 to -92 region has a GC box (GGGGCGG) surrounded by overlapping GGAGG repeats. To analyze nuclear factor binding to this region, we made a series of sequence substitutions in the GGAGG repeats, the GC box, or both regions. Gel mobility shift assays indicated that most of the nuclear factor complexes formed between the wild-type -131/-92 sequence and either HT29 or RINm5F extracts were specific for SP1 or related proteins that recognize a GC box. Mutation of either the GGAGG repeats or the GC box did not eliminate the binding of Sp1 or related nuclear factors, suggesting that both the GGAGG repeats and the GC box could bind Sp1-related factors. Mutation of both these sites eliminated the binding of Sp1-related factors. In the absence of SP1 binding sites, this region had a negative effect on transcription in HT29 and a positive effect on transcription in RINm5F cells. These data support the notion that Sp1 and/or a related factor may control both positive and negative gene expression and suggest that the -131/-92 region may be involved in regulating tissue-specific levels of alpha2A-adrenergic receptor gene expression.

Localization of alpha 2A- and alpha 2B-adrenergic receptor subtypes in brain.

Recent studies have shown that all three subtypes of alpha2-adrenergic receptor (alpha2-AR) are found in brain. The purpose of this study was to map the subtype localization of the alpha2A- and alpha2B-ARs in brain structures. RNase protection shows that both the alpha2A- and alpha2B-ARs are detectable in cortex, cerebellum, pons-medulla, and hypothalamus. We tested probes derived from the alpha2A- and alpha2B-AR cDNAs on cell lines that express each of the alpha2-AR subtypes to establish the subtype specificity of these probes for in situ hybridization. Then we used the alpha2A- and alpha2B-AR probes for in situ hybridization on sagittal and coronal sections of rat brain. Both alpha2A and alpha2B mRNA were detected throughout the brain. Overall, there appears to be a greater expression of message for alpha2A- than alpha2B-AR in most brain areas, with the exception of the thalamus. Developing these probes for in situ hybridization is an important step for further studies on the exact role of the alpha2-AR subtypes in neurons that modulate cardiovascular function.

A negative regulatory element in the promoter region of the rat alpha 2A-adrenergic receptor gene overlaps an SP1 consensus binding site.

Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.

Alpha 2 adrenergic receptor subtypes expressed in Chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21ras independent pathway.

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

Role of the Q10 class I regulatory element region 1 in controlling tissue-specific expression in vivo.

The MHC class I regulatory element (CRE) region 1 has been previously described as a positive cis-acting regulatory element essential for class I gene expression. We have generated transgenic mice (CBA x C57BL/6) with the MHC class I gene H-2Dd driven by two different 400-bp promoter regions of Q10, a nonpolymorphic MHC class I gene expressed in the liver, kidney, and fetal yolk sac. One transgene contained the wild-type Q10 promoter (Q10WT/Dd). The second construct (Q10M3/Dd) had 2 bp substitutions introduced in region 1 of the CRE that reconstituted the CRE inverted repeat present in classical class I genes. Mice containing the wild-type Q10/Dd gene expressed membrane-bound H-2Dd molecules in a tissue-restricted expression pattern similar to that observed for endogenous Q10. In mice containing the mutant construct (Q10M3/Dd), H-2Dd was also expressed in the thymus, a tissue not normally associated with Q10 expression but, surprisingly, the Dd was not expressed in other lymphoid tissues. Furthermore, thymic expression was greatest on double positive (CD4+ CD8+) thymocytes. Thymic Dd expression was correlated with the presence of what appears to be a previously unidentified transcription factor in thymocytes that is capable of interacting with the CRE-inverted repeat. These results show that the mutations in region 1 altered the tissue-specific regulation of the Q10 promoter in vivo, although an intact inverted repeat did not restore the ubiquitous pattern of expression characteristic of classical class I genes. Thus, these results indicate that elements in addition to CRE region 1 in the Q10 promoter region serve to limit ubiquitous tissue expression.

Diverse tissue expression of rat alpha 2-adrenergic receptor genes.

Previously, we have reported two major alpha 2-adrenergic receptor transcripts in rat brain of 3.8 and 3.0 kb and the cloning and characterization of the rat brain complementary DNA (cDNA) (RB alpha 2C) specific for the 3.0-kb messenger RNA. In this report, we used rat brain cDNAs specific for the 3.0 and 3.8 kb transcripts, which encode the alpha 2C- and alpha 2A-adrenergic receptors, respectively, and the RNG alpha 2 cDNA, which encodes for the nonglycosylated alpha 2B-adrenergic receptor in rat, to study tissue-specific expression of the three alpha 2-adrenergic receptor genes in rat. To eliminate cross-hybridization of probes with transcripts from other alpha 2 genes, we subcloned fragments that encode for the highly divergent third cytoplasmic loop of each rat alpha 2-adrenergic receptor cDNA and used RNase protection analysis to detect specific transcripts. We show that the three rat alpha 2-adrenergic receptor genes have diverse patterns of tissue expression, and although transcripts specific for each alpha 2-adrenergic receptor gene are found in brain and kidney, the levels of expression of each subtype differ in these tissues. We speculate on the significance of tissue-specific expression of the alpha 2-adrenergic receptor genes.

Promoter region of the human alpha 2A adrenergic receptor gene.

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.

Cloning and expression of a rat brain alpha 2B-adrenergic receptor.

We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.

Site-specific mutagenesis of the class I regulatory element the Q10 gene allows expression in non-liver tissues.

Classical transplantation Ag are found on nearly all cells, whereas Ag encoded by the genes of the Qa/Tla region have a restricted tissue distribution. To investigate the cause of these different patterns of expression, we have compared the regulatory regions of Q10, a Qa region gene that is expressed only in liver, with those of H-2Ld. Gel retardation analysis shows that the nuclear factor (rI) that binds to the inverted repeat (TGGGGATTCCCCA) of the class I regulatory element (CRE) present in H-2Ld and other classical class I genes does not bind to the equivalent region of the Q10 gene. However, the Q10 CRE binds another nuclear factor (rII) that binds to the H-2Ld CRE. The sequence of the Q10 CRE differs from the sequence present in classical class I genes by three nucleotides, two of these changes are within the inverted repeat sequence (TGaGGAcTCCCCA) and disrupt the region of dyad symmetry. We have used site-specific in vitro mutagenesis to individually change these two nucleotides and have investigated the ability of this region to promote transcription with and without these substitutions. A change of either base restores transcriptional activity in chloramphenicol acetyl transferase assays and allows for binding of the rI nuclear factor. These results suggest that the failure of the Q10 CRE to bind the rI nuclear factor may play a role in preventing Q10 expression in tissues other than the liver and fetal yolk sac. Thus, the dichotomy between the widespread tissue expression of classical class I genes and the restricted tissue expression of class I genes of the Qa/Tla region may be due in part to differences in the cis acting regulatory sequences that interact with trans-acting nuclear factors to direct transcription.

Signals controlling alternative splicing of major histocompatibility complex H-2 class I pre-mRNA.

The use of alternative splice acceptor sites during the removal of intron 7 in pre-mRNA splicing produces two forms of H-2Kb protein: the predominant form, derived from a transcript that has spliced at the upstream splice acceptor site for exon 8 (long exon 8), and a Kb molecule derived from a transcript that has spliced at the downstream acceptor site for exon 8 (short exon 8). We have identified a potential lariat branch point adenosine for the upstream acceptor splice site. This adenosine is found 28 bp from the splice junction and is contained in the sequence AGTGATGG. D-region genes, which use only the downstream splice site, have the sequence AGTGGTGG. We have used in vitro mutagenesis to change this A of the H-2Kb gene to G and have made the reciprocal change in H-2Dd. Elimination of this adenosine in H-2Kb alters the pattern of pre-mRNA splicing and results in a predominance of the Kb molecules with short exon 8 encoded sequences. However, the addition of an adenosine in H-2Dd is not sufficient to direct splicing to the upstream site.

Identification of a human salivary amylase gene. Partial sequence of genomic DNA suggests a mode of regulation different from that of mouse, Amy1.

We report the identification and partial sequence for a human salivary amylase gene, Amy1. The genomic sequence was compared with known human pancreatic and salivary amylase cDNA sequences and another salivary amylase gene sequence. While most of the intron/exon structure and coding sequences are highly similar to other amylase DNAs, the bulk of the 5' end varies significantly. Major differences in the positions and structures of promoters between human and mouse Amy1 genes suggest that there are important differences between the tissue-specific expressions in the two mammals. Some potential enhancer sequences were identified.