cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed.     

Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates

The fraction and DNA composition of simian virus 40 chromosomes that were complexed with large T-antigens (T-Ag) were determined at the peak of viral DNA replication. Simian virus 40 chromatin containing radiolabeled DNA was extracted by the hypotonic method of Su and DePamphilis (Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470, 1976) and then fractionated by sucrose gradient sedimentation into replicating (90S) and mature (70S) chromosomes. Viral chromosomes containing T-Ag were isolated by immunoprecipitation with saturating amounts of either an anti-T-Ag monoclonal antibody or an anti—T-Ag hamster serum under conditions that specifically precipitated T-Ag protein from cytosol extracts. An average of 10% of the uniformly labeled DNA in the 90S pool and 7.5% in the 70S pool was specifically precipitated, demonstrating that under these conditions immunologically reactive T-Ag was tightly bound to only 8% of the total viral chromosomes. In contrast, simian virus 40 replicating intermediates (RI) represented only 1.2% of the viral DNA, but most of these molecules were associated with T-Ag. At the shortest pulse-labeling periods, an average of 72 ± 18% of the radiolabeled DNA in 90S chromosomes could be immunoprecipitated, and this value rapidly decreased as the labeling period was increased. Electron microscopic analysis of the DNA before and after precipitation revealed that about 55% of the 90S chromosomal RI and 72% of the total RI from both pools were specifically bound to T-Ag. Comparison of the extent of replication with the fraction of RI precipitated revealed a strong selection for early replicating DNA intermediates. Essentially all of the RI in the 70S chromosomes were less than 30% replicated and were precipitated with anti—T-Ag monoclonal antibody or hamster antiserum. An average of 88% of the 90S chromosomal RI which were from 5 to 75% replicated were immunoprecipitated, but the proportion of RI associated with T-Ag rapidly decreased as replication proceeded beyond 70% completion. By the time sibling chromosomes had separated, only 3% of the newly replicated catenated dimers in the 90S pool (<1% of the dimers in both pools) were associated with T-Ag. Measurements of the fraction of radiolabeled DNA in each quarter of the genome confirmed that T-Ag was preferentially associated with newly initiated molecules in which the nascent DNA was nearest the origin of replication. These results are consistent with a specific requirement for the binding of T-Ag to viral chromosomes to initiate DNA replication, and they also demonstrate that T-Ag does not immediately dissociate from chromosomes once replication begins. The biphasic relationship between the fraction of T-Ag—containing RI and the extent of DNA replication suggests either that 1 or 2 molecules of T-Ag remain stably bound until replication is about 70% completed or that 4 to 6 molecules of T-Ag are randomly released from each RI at a uniform rate throughout replication.     

Local cerebral glucose utilization in the hippocampus of old rats

Summary The local cerebral glucose utilization (LCGU) was measured in the different areas and layers of the Ammon's horn and dentate gyrus of young adult (3 to 4-month-old) rats, and of 27-month-old rats with proven cognitive deficits. The LCGU was determined by quantitative [¹⁴C]2-deoxyglucose autoradiography. Compared to young animals, in the old rats the LCGU was significantly reduced by 12% to 15% in the oriens layers of CA1 and CA2, the pyramidal layers of the CA sectors 1–3, the radiatum and lacunosum-molecular layers of CA2 and CA3 and in the lucidum layer of CA3. The LCGU values of all the other layers of the Ammon's horn and the dentate gyrus did not differ significantly between young and old rats. The pattern of the LCGU reduction found in the old rats roughly resembles changes found after fimbra-fornix lesions or systemic administration of scopolamine, suggesting a functionally important deficit in the cholinergic innervation of the old rats' hippocampi.     

Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53.

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.     

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.     

Chromatin assembly. Relationship of chromatin structure to DNA sequence during simian virus 40 replication

The accessibility of five specific DNA sequences to six different single site restriction endonucleases was evaluated in replicating and mature simian virus 40 chromosomes isolated by three different methods. Electron microscopic and gel electrophoretic analysis of the DNA digestion products demonstrated that DNA accessibility in chromatin was established within 400 base pairs of replication forks and remained essentially unchanged during production of mature chromosomes and their subsequent re-entry into the replication pool. Saturating amounts of each enzyme reproducibly cut a fraction of the chromosomes, ranging from 13 to 49%. This is consistent with a nearly random phasing of chromatin structure. Examples in which all chromosomes were either cleaved or intact were never observed. Although variation in the accessibility of DNA sites near the origin of replication could be interpreted as preferred phasing in about 25% of the chromosomes, the finding that two isoschizomers, Hpa II and Msp I, did not cut chromosomes to the same extent precludes an unambiguous interpretation of the extents of cleavage of individual restriction enzymes. Since the extent of DNA cleavage observed at each restriction site was essentially indistinguishable in replicating as compared to mature chromosomes, the accessibility of DNA sequences near the origin is not obviously related to replication. Furthermore, the accessibility of DNA sites on one arm of a single replication fork was the same as the homologous sites on the other arm, consistent with a nearly random phasing of chromatin structure on both arms. This suggests that chromatin assembly occurs independently on the 2 sibling molecules of a single replicating chromosome.     

Are Sage-Grouse Fine-Scale Specialists or Shrub-Steppe Generalists?

Sage-grouse (Centrocercus spp.) are influencing rapidly evolving land management policy in the western United States. Management objectives for fine-scale vegetation characteristics (e.g., grass height >18 cm) have been adopted by land management agencies based on resource selection or relationships with fitness proxies reported among numerous habitat studies. Some managers, however, have questioned the appropriateness of these objectives. Moreover, it remains untested whether habitat–fitness relationships documented at fine scales (i.e., among individual nests within a study area) also apply at scales of management units (e.g., pastures or grazing allotments), which are many orders of magnitude larger. We employed meta-analyses of studies published from 1991 to 2019 to help resolve the role of fine-scale vegetation structure in nest site selection and nest success across the geographic range of greater sage-grouse (C. urophasianus) and evaluate the validity of established habitat management objectives. Specifically, we incorporated effects of study design and functional responses to resource availability in meta-regression models linking vegetation structure to nest site selection, and used a novel meta-analytic approach to simultaneously model vegetation structure and its relationship to nest success. Our approach tested habitat relationships at a range-wide extent and a grain size closely matching scales at which agencies make management decisions. We found moderate, but context-dependent, effects of shrub characteristics and weak effects of herbaceous vegetation on nest site selection. None of the tested vegetation characteristics were related to variation in nest success, suggesting nesting habitat–fitness relationships have been inappropriately extrapolated in developing range-wide habitat management objectives. Our findings reveal surprising flexibility in habitat use for a species often depicted as having very particular fine-scale habitat requirements, and cast doubt on the practice of adopting precise management objectives for vegetation structure based on findings of individual small-scale field studies. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.