Methylation and expression of the Myo D1 determination gene

Mouse embryo cells induced to differentiate with the demethylating agent 5-azacytidine represent an excellent model system to investigate the molecular control of development. Clonal derivatives of 10T1/2 cells that have become determined to the myogenic or adipogenic lineages can be isolated from the multipotential parental line after drug treatment. These determined derivatives can be cultured indefinitely and will differentiate into end-stage phenotypes on appropriate stimulation. A gene called Myo D1, recently isolated from such a myoblast line, will confer myogenesis when expressed in 10T1/2 or other cell types (Davis et al. 1987). The cDNA for Myo D1 contains a large number of CpG sequences and the gene is relatively methylated in 10T1/2 cells and an adipocyte derivative, but is demethylated in myogenic derivatives. Myo D1 may therefore be subject to methylation control in vitro. On the other hand, preliminary observations suggest that Myo D1 is not methylated at CCGG sites in vivo so that a de novo methylation event may have occurred in vitro. These observations may have significance in the establishment of immortal cell lines and tumours.     

Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain.

The product of the adenovirus E1A 13S mRNA can both stimulate and repress the expression of certain viral and cellular genes. As with several other regulatory proteins, E1A has a short half-life, approximately 40 min. Although this short half-life is observed in cells expressing the E1A gene, it is not the case with cells injected with E1A protein, where its half-life is very long, generally greater than 15 h. We have sought to reconcile these apparent differences in E1A stability. Using Xenopus oocytes, we find that E1A exhibits its characteristic short half-life when it is synthesized from injected mRNA while it has a very long half-life when it is injected as a protein synthesized originally in Escherichia coli or reticulocyte lysates. In order to delineate the amino acids responsible for rapid E1A turnover, several deletion mRNAs were constructed, injected into oocytes, and E1A half-life determined. Carboxyl-terminal deletions and an internal deletion of residues 38-86 failed to increase the half-life of E1A. In contrast, amino-terminal deletions of 70 and 14 residues resulted in very stable E1A proteins (t1/2 greater than 20 h). Furthermore, deletion of the second amino acid, an arginine, resulted in a stable E1A protein. The amino-terminal region of E1A was able to induce the rapid turnover of a normally stable protein, beta-globin, in oocytes injected with an E1A-globin chimeric mRNA. This E1A-induced instability of globin was abolished, however, when the protein was first synthesized in reticulocyte lysates and then injected into oocytes. The amino-terminal region of E1A is also important in governing halflife in adenovirus-infected HeLa cells. These results demonstrate that the half-life of E1A is established cotranslationally through a mechanism involving sequences within the amino-terminal 37 residues.     

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.     

Gender difference in the relationship of performance in the handgrip and standing long jump tests to lean limb volume in young adults

Groups of young, adult males and females performed the handgrip and standing long jump tests. Their total forearm and leg volumes were calculated from a series of circumference and length measurements, and the lean volumes (bone + muscle) calculated by taking the skinfold thickness into consideration. In the handgrip, the mean female performance was 298 N compared with 496 N for the males. In the standing long jump, mean performance expressed as distance x body mass was 87.3 kg.m for females compared with 137.7 kg.m for males. These superior performances of males could simply reflect their greater muscle mass, as the mean lean volumes of female and male limbs respectively were 0.54 l and 0.89 l for forearms, and 11.82 l and 14.82 l for the two legs. However, when the performances of males and females were grouped by lean limb volume, it was found that while in both tests there were linear relationships, males and females did not share a common line. In both tests the male relationship was at a higher level than the female; therefore, for a given lean volume, the male performance was significantly superior to that of the female. The gender difference found in this study has not been seen in other studies in which the performance of skeletal muscle has been related to the cross-sectional area of the active muscles and the possible reasons for the differences are considered.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Sequential transposition of Tn916 among Staphylococcus aureus protoplasts

Transposition of the Streptococcus faecalis conjugal tetracycline-resistance transposon Tn916 between S. aureus strains occurred when protoplasts of donor and recipient strains were regenerated together without prior fusion. Under these conditions, only Tn916 was transferred; spontaneous fusion of parental protoplasts is therefore unlikely to be responsible for Tn916 transfer. While the exact nature of this transfer remains unclear, it appears to resemble Tn916 conjugal transposition reported in S. faecalis. Evidence for sequential transpositions of Tn916 was obtained by 3-factorial transformation analyses and confirmed by DNA-DNA hybridizations. The ability of Tn916 to transpose within S. aureus and occupy diverse chromosomal sites demonstrates the value of this transposon in genetic studies of S. aureus.