The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.
Parathyroid cells express a cell surface receptor, coupled to the mobilization of intracellular Ca2+, that is activated by increases in the concentration of extracellular Ca2+ and by a variety of other cations. This "Ca2+ receptor" (CaR) serves as the primary physiological regulator of parathyroid hormone secretion. Alterations in the CaR have been proposed to underlie the increases in Ca2+ set-point seen in primary hyperparathyroidism due to parathyroid adenoma. We have isolated human CaR cDNAs from an adenomatous parathyroid gland. The cloned receptor, expressed in Xenopus oocytes, responds to extracellular application of physiologically relevant concentrations of Ca2+ and other CaR agonists. The rank order of potency of CaR agonists displayed by the native receptor (Gd3+ > neomycin B > Ca2+ > Mg2+) is maintained by the expressed receptor. The nucleotide sequence of the human CaR cDNA predicts a protein of 1078 amino acids with high sequence similarity to a bovine CaR, and displays seven putative membrane-spanning regions common to G protein-coupled receptors. The deduced protein sequence shows potential sites for N-linked glycosylation and phosphorylation by protein kinase C and has a low level of sequence similarity to the metabotropic glutamate receptors. Comparison of the cDNA sequence to that of the normal human CaR gene showed no alteration in the coding region sequence of the CaR in this particular instance of parathyroid adenoma. Human cDNA clones with differing 5'-untranslated regions were isolated, suggesting alternative splicing of the parathyroid CaR mRNA. A rare variant cDNA clone representing a 10 amino acid insertion into the extracellular domain was also isolated. Northern blot analysis of normal and adenomatous parathyroid gland mRNA identified a predominant transcript of approximately 5.4 kilobases, and less abundant transcripts of approximately 10, 4.8 and 4.2 kilobases in RNA from the adenoma. While there is no evidence for alteration of the primary amino acid sequence of the CaR in this adenoma, modulation of CaR biosynthesis through alternative RNA processing may play a role in set-point alterations.
Three pro-alpha 1 collagen cDNA clones, pCg1, pCg26, and pCg54, and two pro-alpha 2 collagen cDNA clones, pCg 13 and pCg45, were subjected to extensive DNA sequence determination. The combined sequences specified the amino acid sequences for chicken pro-alpha 1 and pro-alpha 2 type I collagens starting at residue 814 in the collagen triple-helical region and continuing to the procollagen C-termini as determined by the first in-phase termination codon. Thus, the sequences of 272 pro-alpha 1 C-terminal, 260 pro-alpha 2 C-terminal, 201 pro-alpha 1 helical, and 201 pro-alpha 2 helical amino acids were established. In addition, the sequences of several hundred nucleotides corresponding to noncoding regions of both procollagen mRNAs were determined. In total, 1589 pro-alpha 1 base pairs and 1691 pro-alpha 2 base pairs were sequenced, corresponding to approximately one-third of the total length of each mRNA. Both procollagen mRNA sequences have a high G+C content. The pro-alpha 1 mRNA is 75% G+C in the helical coding region sequenced and 61% G&C in the C-terminal coding region while the pro-alpha 2 mRNA is 60% and 48% G+C, respectively, in these regions. The dinucleotide sequence pCG occurs at a higher frequence in both sequences than is normally found in vertebrate DNAs and is approximately 5 times more frequent in the pro-alpha 1 sequence than in the pro-alpha 2 sequence. Nucleotide homology in the helical coding regions is very limited given that these sequences code for the repeating Gly-X-Y tripeptide in a region where X and Y residues are 50% conserved. These differences are clearly reflected in the preferred codon usages of the two mRNAs.
Cloned cDNAs corresponding to mRNAs which accumulate in nitrogen-fixing root nodules of soybean (nodulin mRNAs) were used as probes to investigate the sizes, sequence relationships, tissue specificities and developmental accumulations of individual nodulin mRNA sequences. Northern blot analysis indicated that the NodB, NodC and NodD mRNA sequences are 1 150, 770, and 3 150 nucleotides long, respectively, which is consistent with the previously determined sizes of the hybrid-selected translation products (27 000, 24 000 and 100 000 MW, respectively). The NodA clones pNodA15 and pNodA25 hybridized to two mRNAs of lengths 1 600 and 1 100 nucleotides, indicating that they contain significant sequence homologies. However, increasing the hybridization stringency showed that the pNodA15 clone encodes the 1 600 nucleotide mRNA corresponding to the major NodA hybrid-selected translation product (44 000 MW) while pNodA25 encodes an mRNA of 1 100 nucleotides. The latter probably corresponds to one of two smaller (23 500 and 24 500 MW) in vitro translation products. RNA dot-blot hybridizations indicated that nodulin and leghemoglobin mRNAs began to appear and accumulate in Rhizobium infected root tissue very early (day 3 to 5) and reached fully induced levels by day 11. This accumulation was specific for nodule tissue (except for the NodD sequence) and preceded the accumulation of nitrogen fixation activity. Nodules produced by different effective Rhizobium strains accumulated similar levels of leghemoglobin and nodulin mRNAs while ineffective strains had a pleiotropic affect. While one ineffective strain (61A24) gave reduced levels of all these mRNAs, the other (SM5) gave levels which were nearly normal by the time nitrogen fixation activity should have reached its maximal level (day 17). Thus, leghemoglobin and nodulin genes are switched on soon after infection, prior to nodule morphogenesis, and the switch occurs prior to and is independent of nitrogen fixation activity.
Plant gene products that could play a role in the process of symbiotic nitrogen fixation in leguminous plants were detected by screening a cDNA library prepared from soybean nodule poly(A)+ RNA. About 13% of the 5,700 clones screened contained sequences detectable with a root cDNA probe while about 2,100 clones (37% of the library) contained sequences that were detectable only with nodule cDNA. Five unique sequence species, accounting for more than half of the 2,100 nodule-specific clones, were identified by cross-hybridization experiments. The most abundant species, represented by 860 clones, encodes the wellcharacterized protein, leghemoglobin (Lb). The other four species, designated NodA, NodB, NodC, and NodD, are represented by 350, 55, 61, and 6 clones, respectively. Each of these four species was found to be encoded by the plant nuclear genome at low copy number. The transcripts corresponding to the nodule-specific clones represented 12-15% (Lb), 6% (NodA), and 0.5-1.1%
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