W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of amago salmon (Oncorhynchus rhodurus).

To examine the changes in secretion of growth hormone (GH) and prolactin (PRL) with reference to their osmoregulatory roles, changes in pituitary mRNA levels and plasma concentrations of these hormones were examined during seawater adaptation in silvery juveniles (smolts) and precociously mature males (dark parr) of amago salmon (Oncorhynchus rhodurus). Transfer to seawater increased plasma sodium levels in both smolts and dark parr. Smolts adjusted their plasma sodium to the level associated with seawater-adaptation (165 mEq/liter) within 3 days, whereas no adjustment was seen in dark parr; the latter failed to survive in seawater for more than 3 days. In smolts, plasma GH levels increased significantly 1 day after transfer, whereas there was no significant change in dark parr. An increase in GH mRNA levels was observed in smolts in association with increased plasma GH, whereas there was no change in dark parr. In contrast, a reduction in plasma PRL levels was consistently observed in both smolts and dark parr after transfer to seawater. However, there was no significant change in PRL mRNA levels in either smolts or dark parr. These results suggest that both gene expression and release of GH are activated by seawater transfer only in smolts with adequate seawater adaptability, whereas PRL gene expression is decreased after seawater transfer regardless of seawater adaptability.     

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10⁶ cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Platelet talin is phosphorylated by calyculin A

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Prevalence of feline viral antibodies in random-source laboratory cats

Over a period of 1973 to 1979, a serologic survey of virus infections was conducted on feline sera collected in four universities which located in different prefectures; Obihiro, Saitama, Kanagawa and Tokyo. A significant hemagglutination-inhibition (HI) antibody titer of 1 : 8 or higher to feline panleukopenia virus (FPLV) was detected in 130 (58%) of the 226 sera used. No remarkable difference in the HI antibody prevalence in cats to FPLV was recognized by years or localities. Of a total of 188 cats tested, 99 (53%) presented positive serum neutralizing (SN) antibody titers to the No. 1 strain of feline calicivirus (FCV). Especially in Kanagawa, 17 (77%) of the 22 cats had positive SN titers. However, only 42 (22%) of the 188 sera showed positive SN titers to the Kyoritsu strain of FCV. Such lower positivity in the cats was observed with 13% in the SN test to human reovirus type 3 (Reo-3). The incidence of positive SN antibodies to feline rhinotracheitis virus (FRV) also remained in low values of 20 to 27% with the exception of high percentage of 86 in Tokyo. The dissemination of FPLV, FRV, FCV and Reo-3 was briefly discussed in relation with the age distribution of viral antibodies in cats.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Improvement of Intracellular Traffic System by Overexpression of KDEL Receptor 1 in Antibody‐Producing CHO Cells

The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C‐terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven‐transmembrane protein that cycles between the ER and cis‐Golgi to capture missorted KDEL chaperones from post‐ER compartments in a pH‐dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody‐producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production.