Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.     

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.     

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific puffs in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5×10^–6 mol/l 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca²⁺-dependent increase of nuclear volume by 23.0±2.3%. Amiloride, a blocker of plasma membrane Na⁺/H⁺ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9±3.9%. Measurments of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23±0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amilorde-sensitive plasma membrane Na⁺/H⁺ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.     

The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

The human complement component C8B gene: structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (, and ), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the -subunit is non-covalently bound to the disulfide-linked - subunit. Using a full-length C8 cDNA probe, we isolated several clones from human genomic DNA libraries. Four clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were shotgun subcloned into M13. C8-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.     

Climate change disrupts core habitats of marine species

Driven by climate change, marine biodiversity is undergoing a phase of rapid change that has proven to be even faster than changes observed in terrestrial ecosystems. Understanding how these changes in species composition will affect future marine life is crucial for conservation management, especially due to increasing demands for marine natural resources. Here, we analyse predictions of a multiparameter habitat suitability model covering the global projected ranges of >33,500 marine species from climate model projections under three CO₂ emission scenarios (RCP2.6, RCP4.5, RCP8.5) up to the year 2100. Our results show that the core habitat area will decline for many species, resulting in a net loss of 50% of the core habitat area for almost half of all marine species in 2100 under the high-emission scenario RCP8.5. As an additional consequence of the continuing distributional reorganization of marine life, gaps around the equator will appear for 8% (RCP2.6), 24% (RCP4.5), and 88% (RCP8.5) of marine species with cross-equatorial ranges. For many more species, continuous distributional ranges will be disrupted, thus reducing effective population size. In addition, high invasion rates in higher latitudes and polar regions will lead to substantial changes in the ecosystem and food web structure, particularly regarding the introduction of new predators. Overall, our study highlights that the degree of spatial and structural reorganization of marine life with ensued consequences for ecosystem functionality and conservation efforts will critically depend on the realized greenhouse gas emission pathway.     

NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.