Stabilization of eukaryotic initiation factor 4E binding to the mRNA 5'-Cap by domains of eIF4G.

The eukaryotic cap-binding complex eIF4F is an essential component of the translational machinery. Recognition of the mRNA cap structure through its subunit eIF4E is a requirement for the recruitment of other translation initiation factors to the mRNA 5'-end and thereby for the attachment of the 40 S ribosomal subunit. In this study, we have investigated the mechanistic basis of the observation that eIF4E binding to the cap is enhanced in the presence of the large eIF4F subunit, eIF4G. We show that eIF4E requires access to both the mRNA 5'-cap and eIF4G to form stable complexes with short RNAs. This stabilization can be achieved using fragments of eIF4G that contain the eIF4E binding site but not the RNA recognition motifs. Full-length eIF4G is shown to induce increased eIF4E binding to cap analogues that do not contain an RNA body. Both results show that interaction of eIF4G with the mRNA is not necessary to enhance cap binding by eIF4E. Moreover, we show that the effect of binding of full-length eIF4G on the cap affinity of eIF4E can be further modulated through binding of Pab1 to eIF4G. These data are consistent with a model in which heterotropic cooperativity underlies eIF4F function.

Examination of methods for the deep freezing of goat semen.

In four experiments, freezing of goat semen in straws was examined and compared with conventional pellet-freezing. The optimum freezing regime was attained by holding straws in vapour 4 cm above liquid nitrogen for 30 s or longer, followed by plunging into liquid nitrogen. Straws exposed to vapour for only 10 s did not cool sufficiently before plunging, and cell viability was seriously impaired. Initiation of ice crystallization and the pattern of cooling of semen depended on size of straws and the type of rack used to hold straws in vapour during freezing. Nevertheless, semen was equally well stored in straws of 0.25 mL and 0.50 mL capacity and straws were most efficiently filled at 30 degrees C. Modified Tris media were used to dilute semen at rates (semen: diluent) of 1:2, 1:5, 1:11 and 1:23. Spermatozoa survived best in hypertonic diluents and deteriorated during post-thawing incubation in media of low tonicity, whilst the optimum diluent depended on rate of extension before freezing. The cooling curves differed markedly for semen frozen in straws in liquid nitrogen vapour and pellets on dry ice. Viability of spermatozoa was better for the pellet-frozen semen.

Artificial insemination of Cashmere goats: effects on fertility and fecundity of intravaginal treatment, method and time of insemination, semen freezing process, number of motile spermatozoa and age of females.

The experiments, using a total of 1833 Cashmere does, were conducted to examine the effects on fertility and fecundity of intravaginal treatment for control of ovulation, time of insemination by cervical and laparoscopic methods, semen processing in pellets and straws, and number of frozen-thawed motile spermatozoa. Adult does (greater than or equal to 2.5 years) that had previously kidded were inseminated into the cervix, and maiden (kid: 6-8 months; hogget: 18 months) and adult does were inseminated laparoscopically. The rates of pregnancy determined by ultrasonic scanning (75-80 days after insemination) for cervical (Experiment 1) and laparoscopic (Experiments 2 and 3) insemination were 39.1%, 63.6% and 52.1%. The number of motile spermatozoa did not affect fertility after cervical (80, 120 and 160 x 10(6) or laparoscopic (Experiment 2: 15, 30 and 60 x 10(6); Experiment 3:5, 10 and 20 x 10(6) insemination. The method of insemination or number of motile spermatozoa did not affect fecundity. With cervical insemination, fertility and fecundity improved with increasing depth of insemination into the reproductive tract. Intravaginal sponges containing fluorogestone acetate and controlled internal drug release (CIDR) devices containing progesterone were equally effective for control of ovulation when combined with an injection of pregnant mare's serum gonadotrophin (PMSG; 200 i.u.) before insemination. Fertility was higher when does were inseminated before than after ovulation. After laparoscopic insemination, results were similar for semen frozen as pellets and in straws, and for 3-, 6- and 24-fold pre-freezing dilution of semen. For kid, hogget and adult does in Experiment 3, pregnancy rates were 34.0%, 55.4% and 63.9% and rates of fecundity were 1.17, 1.22 and 1.27 fetuses/pregnant doe.