Molecular cloning and chromosomal localization of the human homologue of a B-lymphocyte specific protein tyrosine kinase (blk).

A cDNA encoding the human homologue of the murine protein tyrosine kinase, blk, has been cloned from a human B-lymphocyte cDNA library by cross-species hybridization using the murine blk cDNA as a probe. The sequence of the 2608 bp human blk cDNA clone contains an open reading frame encoding a predicted 505 amino acid protein with SH3, SH2 and catalytic domains that contain consensus sequences of the src protein tyrosine kinase family. Comparison of human and murine blk sequences indicated that they share 86% amino acid identity, the most conserved region being the catalytic domain (93% identity). Like the murine blk gene human blk is expressed only in B lymphocytes. The human blk gene was mapped to chromosome 8 at p22-23.

The regulation of uterine tissue factor by estrogen.

Tissue factor (TF) is a transmembrane protein that initiates coagulation and indirectly catalyzes the conversion of prothrombin to thrombin. We previously showed that treatment of immature rats with estradiol (E(2)) stimulated a rapid increase in TF mRNA and protein in the uterus. Our current experiments usingin situ hybridization show that the increase in TF mRNA occurred primarily in the stromal cell layer. The effect of E(2) to increase TF mRNA occurred in uterine organ cultures but not in separated epithelial and stromal cellsin vitro. Thrombin and the phorbol ester, TPA, compounds which regulate TF expression in other cell types by activation of protein kinase C (PKC), increased TF mRNA in both uterine organ cultures and in separated uteriné cells. The 5' regulatory region of the TF gene was examined for the presence of an estrogen response element (ERE) using a plasmid, pTFCAT, containing -740 to + 15 bp of the mouse TF promoter upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. There was no response to E(2) in HeLa cells cotransfected with pTFCAT and a human ER construct, pHEO. In contrast, E(2) increased CAT activity in cells cotransfected with a positive-control plasmid, containing the consensus ERE cloned upstream of the thymidine kinase promoter-driven CAT gene, and pHEO. CAT activity was also increased by TPA in cells transfected with pTFCAT. In summary, E(2) induces TF mRNA in uterine organ culture indicating that systemic factors are not absolutely required for the effect. However, E(2) injection induces transudation of plasma prothrombin into the uterus where it may be converted to thrombin. Thus thrombin may contribute to E(2)-induction of TF mRNAin vivo. An ERE was not identified in the 750 bp immediately 5' to the transcription start site of the TF gene although a TPA-responsive element was present. It is postulated that E(2) may induce TF mRNA by multiple indirect pathways including stimulation of PKC and Jun and Fos transcription factors, and by generation of thrombin in the uterus.

Significance of lumbosacral list and low-back pain. A controlled radiographic study.

A prospective study of 43 patients with low-back pain was performed to determine if there exists 1) a significant correlation between lumbosacral list and low-back pain, 2) a significant difference between measured list in patients pretreatment and posttreatment, and/or 3) a significant difference in results obtained using two different measuring techniques. All patients had standing antero-posterior radiographs. The radiographs were analyzed for lumbosacral list using two measurement methods. The results indicated that there was no significant correlation between lumbosacral list and low-back pain and no significant change in measured list in individuals pretreatment and posttreatment. There was a significant difference between the two methods described above. The intercristal line (ICL) method proved most precise.

A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor.

Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. We report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. Our results raise the possibility that mTF may also play a role in cell growth.

Melatonin profile in marmots: the influence of catecholamines, hibernation, and light.

The aim of this study was to determine the effects of circulating catecholamines and light on the daily melatonin rhythm in the marmot. Endogenous levels of circulating catecholamines and plasma melatonin were measured during arousal from hibernation in light and studies were performed on the circadian melatonin rhythm in two photoperiods (LD 4:20 and LD 8:16). In addition, studies were done on the capacity of broad-band white light at normal room intensities (32 muW/cm2 or 108 Ix) and of low-intensity monochromatic green light (500 nm; 1.4muW/cm2 or 3.1 Ix) to suppress high nocturnal melatonin levels. We conclude that high levels of plasma catecholamines that occur during arousal from hibernation do not influence the production and secretion of pineal melatonin. During the nocturnal portion of its light/dark cycle, the marmot plasma melatonin rhythm is suppressed by both white light and low-intensity green light.

Earthquake hazards on the cascadia subduction zone.

Large subduction earthquakes on the Cascadia subduction zone pose a potential seismic hazard. Very young oceanic lithosphere (10 million years old) is being subducted beneath North America at a rate of approximately 4 centimeters per year. The Cascadia subduction zone shares many characteristics with subduction zones in southern Chile, southwestern Japan, and Colombia, where comparably young oceanic lithosphere is also subducting. Very large subduction earthquakes, ranging in energy magnitude (M(w)) between 8 and 9.5, have occurred along these other subduction zones. If the Cascadia subduction zone is also storing elastic energy, a sequence of several great earthquakes (M(w) 8) or a giant earthquake (M(w) 9) would be necessary to fill this 1200-kilometer gap. The nature of strong ground motions recorded during subduction earthquakes of M(w) less than 8.2 is discussed. Strong ground motions from even larger earthquakes (M(w) up to 9.5) are estimated by simple simulations. If large subduction earthquakes occur in the Pacific Northwest, relatively strong shaking can be expected over a large region. Such earthquakes may also be accompanied by large local tsunamis.

The simian virus 40 minimal origin and the 72-base-pair repeat are required simultaneously for efficient induction of late gene expression with large tumor antigen.

We have studied the temporal regulation of simian virus 40 (SV40) late gene expression by construction and transient expression analysis of plasmids containing the transposon Tn9 chloramphenicol acetyltransferase gene placed downstream from the late control region. The SV40 origin region in the early (but not the late) orientation promotes chloramphenicol acetyltransferase gene expression efficiently in monkey cells lacking large tumor (T) antigen. In monkey cells producing T antigen, the promoter activity of the late control region is induced by approximately 1,000-fold above the basal level. By deletion and point mutagenesis, we define two domains of the late control region required for efficient induction with T antigen. Domain I is the minimal replication origin containing T-antigen binding site II. Domain II consists of the 72-base-pair (bp) repeat and a 19-bp downstream sequence up to nucleotide 270. Domains I and II should act synergistically because the absence of either one or the other decreases induction efficiency by 2 orders of magnitude. Though a complete copy of domain II is optimal, the origin-proximal 22-bp portion of this domain is sufficient. The 21-bp repeat, located between domains I and II, is dispensable for this induction, as are sequences located downstream from nucleotide 270 in the late orientation.

Mapping of the late promoter of simian virus 40.

Mapping of the simian virus 40 (SV40) late promoter was carried out in the absence of the viral early protein, large tumor (T) antigen, and replication of the viral DNA template. SV40 late control region DNA fragments, containing specific deletions, were cloned directly upstream from the coding region of the herpes simplex virus-1 (HSV-1) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The promoter activities of the fragments were determined by measuring the tk transformation frequencies of the chimeric tk constructs in mouse L TK- APRT- (adenine phosphoribosyltransferase-negative) and human 143B TK- cell lines. The following results were obtained. (i) The SV40 control region functions with equal efficiencies in the early and late promoter orientations. (ii) A major late control element has been localized within the G+C-rich 21-base-pair (bp) repeat. Thus, in conjunction with our earlier results, the 21-bp repeat is a bidirectional promoter element functioning as a major component of both the early and late promoters and is an element that enhances the replication efficiency of SV40 DNA. (iii) Minor late promoters have been localized within the minimal replication origin and the 72-bp repeat. (iv) The minimal replication origin is not per se a constituent of the major late promoter; however, both the minimal replication origin and the 21-bp repeat are required for obtaining high levels of late gene expression observed at late times after infection by SV40. (v) The 72-bp repeat exerts a 4- to 5-fold enhancement of late promoter expression.

SV40 deletion mutants lacking the 21-bp repeated sequences are viable, but have noncomplementable deficiencies.

We have constructed deletion mutants of simian virus 40 (SV40) lacking the two tandemly repeated copies or all three copies of the 21-bp repeated sequence located in the origin region. The mutants were viable, but had lower infectivities compared to the wild type. The mutant lacking two copies of the 21-bp repeat grew fairly well indicating that the one copy of the 21-bp repeat it contains is adequate. The other mutant lacking all the three copies of the 21-bp repeat was also viable but grew poorly. The viability of this mutant suggests that the upstream 72-bp repeated sequence compensates, though only partially, for the absence of the 21-bp repeat. The growth deficiencies of the deletion mutants could not be overcome by complementation with temperature-sensitive helper mutants providing either the early or the late functions of the virus, suggesting that the deficiencies lie in both early and late gene expression and/or in replication.

Territorial limits and functional anatomy of the simian virus 40 replication origin.

The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.