Biomedical subjects
Publications and source records attributed to J Pelletier.
Poliovirus RNA is naturally uncapped, therefore its translation must proceed via a cap-independent mechanism. Translation initiation on poliovirus RNA occurs by binding of ribosomes to an internal sequence within the 5' noncoding region. This novel mechanism of initiation may explain the disparate translation of several other eukaryotic messenger RNAs.
The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirus allows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to a reporter gene and transfected into cells. There was extensive augmentation of the expression of this gene by poliovirus-mediated inhibition of cap-dependent protein synthesis. That the construct initiated in a cap-independent manner was verified through in vitro experiments. Small lesions throughout region P blocked its initiation function, implying that a coherent functional unit, hundreds of nucleotides long, is responsible for cap-independent initiation by poliovirus RNA.
Preparations of either crude or purified protein synthesis initiation factors, when tested by crosslinking to the m7G-cap structure of mRNAs, exhibit specific crosslinking to an 80-kDa protein. Polyclonal antibodies specific for eIF-4B precipitate the 80-kDa cap-radiolabeled protein, thereby demonstrating that eIF-4B binds mRNA near its 5'-terminus.
This study was undertaken to assess the influence of photoperiod on growth hormone (GH) secretion in rams and its possible influence on body weight. Twenty young adult rams were divided into two groups. One was subjected to an annual (AR) and the other to a semestral (SR) light regime during the same 18-month period. In both groups, daylength (DL) varied gradually between 8 to 17 hr. Plasma prolactin (PRL) and GH profiles consisting of 6 hr samples were determined and animals were weighed throughout the course of the experiment. Maximal PRL secretion was observed with largest DL. In contrast, GH secretion increased during increasing DL but it began to decrease before maximal DL was reached in both light regimes. Mean GH secretion was maximal when the DL was about 11 hr in SR and between 8 to 12 hr in AR. Similarly, body weight increased when DL increased and plateaued during decreasing DL in both AR and SR animal groups. Significant (P less than 0.05) differences were observed throughout the course of the experiment according to the effects of decreasing or increasing DL in each group. Analysis of variance showed that the effect of DL on plasma PRL and GH levels and weight velocity (WV) was significant (P less than 0.05) in both light regimes. This suggests that in SR, plasma PRL and GH levels and WV vary according to a six month period.(ABSTRACT TRUNCATED AT 250 WORDS)
The purpose of this study was to examine the relationships between testis weight and the luteinizing hormone (LH) and testosterone releases in rams subjected to 14 successive 2-mo artificial light cycles in which daylength increased from 8 to 16 h in one month and decreased from 16 to 8 h the following month. Testis weights were measured fortnightly. Serial bleedings were performed during 6 to 8 h the last three 2-mo light cycles, when daylengths were 8, 12 (increasing), 16 and 12 h (decreasing) and plasma LH and testosterone were measured by radioimmunoassay. The overall mean testis weight, continuously high and equal to 314 g per testis, was not correlated with daylength. Mean LH levels and LH pulse height varied significantly according to daylength (P<0.05 and P<0.01, respectively) and were maximal on short days (8 h). Both mean and maximal testosterone levels presented variations according to daylength (P=0.05). LH and testosterone pulsatility were quite high (equivalent to 7 to 10.3 pulses/day); however, mean plasma testosterone levels remained low. It is proposed that in rams under 2-mo light cycles, frequent but short stimulation of LH release by decreasing daylength phases allows maximal testis weight, while the interruption of LH stimulation by increasing daylengths prevents overstimulation of testosterone that can inhibit the LH release by negative feedback. Rams a under 2-mo light regimen presented a persistent high testis weight, indicating that seasonality had been broken down in the Ile-de-France male which is normally a marked seasonal breeder.
The control of seasonal reproductive activity in sheep and goats in open sheds, needs extra-light (E) during the photosensitive phase (equivalent to long days, LD), followed by treatment with melatonin (equivalent to short days, SD). In autumn-born Ile-de-France or Lacaune ram lambs, 2 months of E followed by decreasing daylength for 90 days, advanced onset of the first breeding season by allowing males to reach their maximum testis volume and sperm production earlier than for untreated ram lambs. Substitution of decreasing daylength with melatonin implants allowed a transient increase in testis volume. Adult Ile-de-France rams maintained under short light rhythms with 2 month-period, demonstrated, during at least 2 consecutive years, a testis volume equivalent to that observed during the normal breeding season. These light-treated rams produced, during non-breeding season, spermatozoa in the same quantity and quality as during the normal breeding season. In anovulatory out-of-season dairy goats, E treatment was demonstrated to be necessary before melatonin treatment and melatonin to be necessary after E treatment to stimulate oestrous and ovulatory activities. Stimulation of the anovulatory females by the introduction of treated males ("male effect"), appeared to be necessary to obtain maximum stimulation of the treated females. Two months of E, followed by melatonin treatment (daily injection or drenching or subcutaneous implants) allowed cycles with ovulation to be maintained for more than 2 months. Although effective for control of out-of-season reproductive activity, melatonin slightly decreased milk production when applied soon after kidding. So, total control of reproduction in sheep and goats by manipulation of photoperiod in open sheds and melatonin treatments appears feasible in both sexes.
The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.
Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.
Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.
The effect of castration on pituitary androgen and estrogen nuclear receptors was examined in eleven 3-year-old 'Préalpes du Sud' rams which were either intact (N = 5) or surgically castrated (N = 6) 11 months before. To avoid a receptor loss, pituitaries were frozen less than 1 min after slaughter. Specific androgen and estrogen bindings were measured upon extracts from purified nuclei. Neither the concentration of estrogen receptors (30.5 +/- 3.0 vs 28.1 +/- 3.0 fmol/mg protein) nor the affinity constant (ka = 1.21 X 10(9) mol-1 in both groups) differed between intact and castrated rams. Conversely, androgen receptor concentrations differed markedly between groups and were found significantly higher in intact rams (11.3 +/- 1.1 fmol/mg protein) than in castrated rams where nuclear receptors were undetectable in 5 out of 6 animals. This result keeps open the possibility that the decrease of LH sensitivity to testosterone negative feedback observed after long-term castration, could be related to the quasi absence of nuclear receptors, at least at the pituitary level.
Seasonality of the reproductive cycle in sheep is a general phenomenon for mid-latitude breeds. The proximal part (breeding season) and also partially distal part (end of gestation and beginning of lactation) of this cycle is controlled by photoperiod, whatever the form of light regimens. Data are presented which indicate that male and female do not necessarily have the same photoperiodic sensitivity. Gonadal stimulation in the ram starts 1.5-2 months earlier than in the ewe under annual variations. Photoperiod controls the reproductive cycle by the intermediary of the hypothalamo-pituitary axis. There are both a steroid-independent and a steroid-dependent effect of light, depending on both decreasing and increasing daylength in mid-latitudes. Data are also presented which support Bunning's hypothesis on photoperiodic time measurement in mammals. Sheep measure photoperiodic time by using a circadian rhythm of photosensitivity. Daylength is not measured by the total duration of exposure to light but by the illumination of two special set points during the day, one of them entraining the circadian rhythm of photosensitivity and the other inducing or not inducing a physiological response if it is coincident, or not coincident, with photoinducible phase of that rhythm. A photoinducible phase has been found for prolactin secretion, and perhaps also for LH secretion. Melatonin secretion is used by sheep for measuring daylength. However, that secretion disappears during two set points during the day, thus raising the possibility of using alternatively melatonin and light pulse for controlling the reproductive cycle in sheep.
The reovirus S1 gene codes for two polypeptides: sigma 1 and sigma s. In order to characterize the structure and function of the sigma 1 polypeptide, we have expressed the sigma 1 protein in Escherichia coli. The S1 gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expression vector containing the tac (trp-lac) promoter designed to express foreign gene products in E. coli efficiently. The hybrid plasmids, upon induction with isopropyl-beta-D-thiogalactopyranoside, expressed two polypeptides that were detected by [35S]methionine labelling. One of the induced proteins had a relative molecular mass (Mr) of approx. 46,000 and corresponded to sigma 1, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against sigma 1. The second induced protein had a Mr of approx. 12,000 and was very similar to sigma s as judged by comparative tryptic peptide map analysis. Protein sigma 1 produced in E. coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.
Translation initiation in eukaryotes is a complex process involving many factors. A key step in this process is the binding of mRNA to the 43S preinitiation complex. This is generally the rate-limiting step in translation initiation and consequently a major determinant of mRNA translational efficiency. The primary and secondary structure of the mRNA 5' noncoding region have been implicated in modulating translational efficiency. Translational efficiency was shown to be inversely proportional to the degree of secondary structure at the mRNA 5' noncoding region. Furthermore, it was shown that cap-binding proteins that interact with the 5' cap structure (m7GpppN) of eukaryotic mRNAs are involved in the "unwinding" of the mRNA secondary structure, in an ATP hydrolysis mediated event, to facilitate ribosome binding. Thus, cap-binding proteins can potentially regulate mRNA translation. Here, we discuss the available data supporting the notion that eukaryotic 5' mRNA secondary structure plays an important role in translation initiation and the possible regulation of this process.
Ile-de-France ewes were ovariectomized during anoestrus or the mid-luteal phase of an oestrous cycle (day of ovariectomy = Day 0). In a short-term study, FSH concentrations were measured in blood samples collected hourly the day before and on Days 1, 3, 7 and 15 after ovariectomy (10 ewes per group). FSH concentrations increased significantly from 6.1 to 16.5 ng/ml within 1 day of ovariectomy and increased further to 47.1 ng/ml by Day 15. Differences between seasons of ovariectomy were not significant. In a long-term study, FSH concentrations were measured in blood samples collected hourly on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy in anoestrus or the breeding season (10 ewes per group). Further samples were taken (5 ewes/group) at 240 and 365 days after ovariectomy. The pattern of change in FSH after ovariectomy differed between the two seasons and the interaction between season and sampling day was significant. For ewes ovariectomized during anoestrus, FSH concentrations increased to a maximum by Day 180 and remained high thereafter. In contrast FSH increased more slowly in ewes ovariectomized in the breeding season and differences between the groups were significant from Day 90 to Day 270. However, both groups had similar FSH concentrations at Day 365. These results show that FSH concentrations increase rapidly after ovariectomy. There are seasonal differences in FSH concentrations in the absence of ovarian feedback with increases in FSH concentration around the time of the onset of the breeding season. Once FSH concentrations had reached a maximum, major seasonal changes were no longer apparent.(ABSTRACT TRUNCATED AT 250 WORDS)
Two groups of 12 adult Ile-de-France rams were exposed to artificial 6-month light cycles in which daily illumination was provided in one or two photofractions. In Group I, daylength increased linearly from 8 to 16 h in 3 months and decreased similarly from 16 to 8 h. The daily increment or decrement (5.33 min/day) was constant. In Group II, 8 h of light were given in two parts: the main one, 7 h, began at the time of dawn in Group I and an additional 1 h light pulse was coincident with the last hour of the former group. The onset of the pulse changed therefore each day and the interval between dawn of the first block and dusk of the second block of light increased from 8 to 16 h in 3 months and declined from 16 to 8 h the next 3 months. Testicular weight was estimated by an orchidometer every 2 weeks for 2 (N = 12/group) or 3 consecutive light cycles (N = 6/group). The testicular weight variations were identical in both groups. In the 6 rams of each group studied during 3 light cycles, variations of testicular weight were submitted to an harmonic regression analysis according to time and the computed values for the mean, amplitude, period and phase were, respectively, 260 g, 66 g, 185 days and 120 days in Group I and 262 g, 65 g, 181 days and 111 days in Group II. Analysis performed for each ram gave very similar values in all individuals.(ABSTRACT TRUNCATED AT 250 WORDS)
An experiment was conducted to measure LH pulse frequencies in bilaterally adrenalectomized Ile-de-France ewes during the mid-anoestrous season. Seven ewes were adrenalectomized under general anaesthesia and maintained on daily injections of 3 mg deoxycorticosterone acetate (DOCA). Adrenalectomy did not affect the patterns of LH release and the mean intervals between pulses in the adrenalectomized and sham-operated control ewes were 48 and 52 min respectively. Small implants of oestradiol significantly reduced the frequency of LH pulses in both groups and, in the presence of oestradiol, there were no differences in LH release between adrenalectomized and sham-operated ewes, with the mean interpulse intervals being 91 and 89 min respectively. In a second experiment, designed to assess the effects of DOCA on LH release, the mean interpulse intervals in unoperated control ewes (46 min) and unoperated ewes given daily injections of 3 mg DOCA (47 min) were similar to those observed in adrenalectomized and sham-operated ewes. In the presence of small implants of oestradiol, the combination of DOCA and oestradiol decreased the pulse frequency significantly more than oestradiol alone (mean interpulse intervals of 85 and 65 min respectively) suggesting that DOCA may act as a progestagen in sheep. Thus alternative treatments to DOCA for the maintenance of adrenalectomized sheep must be found for future studies on the role of adrenal steroids in the reproductive system. It appears that adrenal steroids do not play a major role in the seasonal changes in LH release in the ewe.
Studies of sheep were undertaken to determine the effects of photoperiod and protein diet on growth hormone (GH) secretion. Rams were subjected to either a control (RI) or an inverted (R2) 6-month (semestral) light regime. In both light regimes day lengths varied gradually between 8 and 16 hr. Within each light regime group of animals, the rams received either a low (L) or a high (H) protein diet containing the same level of energy. Plasma GH profiles consisting of 13 hourly samples were determined at regular intervals corresponding to known day lengths. Analysis of variance indicated that there was a significant effect of day length (P less than 0.01) and protein diet (P less than 0.05) on GH secretion, the two light regimes R1 and R2 were equivalent with respect to GH secretion, and there were no interactions among the three experimental factors. Although mean GH secretion was consistently higher in L groups than in H groups, there was a similar trend in all the animals of increasing GH secretion as day length increased. GH secretion was maximum when the day length reached 13 hr 20 min in increasing photoperiods in L groups (15.6 +/- 1.6 ng X h X ml-1) and 16 hr in H groups (13.0 +/- 1.2 ng X h X ml-1). From these results we conclude that both an increasing day length and a deficiency in protein diet stimulate GH secretion in rams but the GH response to these two factors may involve different regulatory processes and may have different functions.
European breeds of rams appear to be responsive to photoperiodic changes even though there are large differences between breeds in the timing and amplitude of endocrine (LH and testosterone) and gametogenetic variations before the sexual season. Light regimens such as 6-month light cycles or alternations of constant short and long days every 12-16 weeks are able to entrain the parameters of sexual activity. In these regimens in which the period of the light cycle is shortened, LH release is markedly stimulated during decreasing daylength and evidence is presented, from the relationship of LH and testosterone patterns, that the dampening of LH stimulation could simply result from the effect of steroid feedback. However, there is a gap of several weeks between the maximum LH and testosterone concentrations during which testis growth occurs. Experiments were conducted with Ile-de-France rams, markedly seasonal breeders, in which the period of the light cycle was decreased, in different groups of animals, from 6 to 4, 3, 2 or 1 month. Rams submitted to the three light regimens with the longest periods presented testicular variations which paralleled those of the photoperiod, but those kept in the two regimens with the shortest periods had a progressive increase in testicular weight up to the maximum value (300-350 g) with no further major changes. Therefore, in rams kept in 2-month light cycles, testicular weight remained constant for twelve successive cycles (2 years). LH and testosterone plasma measurements indicated that LH was sufficiently stimulated to maintain testicular development during each decreasing daylength phase but that the stimulation was shifted before testosterone could reach levels at which feedback effects could be exerted. However, all the measures of sperm production were at values characteristic of the sexual season. Similar testicular weight maintenance was also obtained in rams submitted to a regimen in which short days (8L:16D) alternated every month with a split photoperiod interpreted as a long day (7L:8D:1L:8D). It is concluded that short light cycles are able to induce persistent reproductive activity in Ile-de-France rams, which may have practical applications in sheep production systems.