Studies on tRNA adaptation, tRNA turnover, precursor tRNA and tRNA gene distribution in Bombyx mori by using two-dimensional polyacrylamide gel electrophoresis.

Eighteen out of twenty amino acids have been used for identifying tRNAs from the silkworm Bombyx mori L. fractionated on two-dimensional polyacrylamide gel electrophoresis. 43 spots out of 53 have been identified. This mapping confirms previous results and brings new answers to some questions on the regulation of tRNA biosynthesis. 1. In addition to quantitative adaptation of tRNAs to the composition of silk proteins (fibroin from the posterior silk gland, sericin from the middle part) and of iso-tRNAs from posterior silk gland to the major codons of fibroin mRNA, we also observe adaptation of tRNA from various tissues to the average amino acid content of proteins from fat body, gut, gonads and carcass of the silkworm. 2. In the silk gland, turnover rates of several tRNA species are similar. The selective accumulation of tRNAs needed for decoding fibroin and sericin mRNAs which takes place during the Vth larval instar, cannot be explained by the occurrence of a preferential degradation of some tRNA species. 3. Under given conditions for incubating silk glands, it is possible to obtain an accumulation of precursor tRNA species, which are enriched in pre-tRNAAla and pre-tRNAGly in the posterior silk gland and pre-tRNASer in the middle part. 4. The distribution of tRNA genes is not random. tRNA genes for glycine, alanine and serine are prominent. Selective transcription of batteries of iso-tRNA genes could explain our data.

Does quantitative tRNA adaptation to codon content in mRNA optimize the ribosomal translation efficiency? Proposal for a translation system model.

Neither a dynamic nor an energetic approach of the translation process has taken into account that intracellular levels of iso-tRNA species are adapted or adjusted to the codon frequency of mRNA being decoded (Bombyx mori silk gland, rabbit reticulocyte). A critical study of available experimental data suggests that the average elongation rate of a protein is maximized in the presence of an adapted tRNA population, usually an homologous tRNA. In addition, the amount of synthesized protein parallels that of corresponding mRNA. Other evidences--including in vitro and in vivo elongation assays with fibroin mRNA--show that individual elongation rates are not uniform. Pauses occur at certain sites of the mRNA chain. The relative lifetime of these pauses depends on the tRNA pool used. Finally, it appears that translation accuracy also depends on the balanced tRNA population. We propose to explain these different effects by using a codon-anticodon recognition model, called "trial and error system" based on a stochastic processing of the ribosome. Accordingly, various acylated tRNA species which surround a ribosome randomly encounter the receptor A site. Every trapped tRNA species is tested for a proper pairing with the codon to be recognized at the level of a comparator or discriminator function. If the pairing is correct, transpeptidation becomes irreversible. If not, the aminoacyl-tRNA is rejected and another randomly trapped tRNA is processed in turn. Mathematical analysis of this model shows that the mean number of trials used for translating the whole sequence of a mRNA is minimized when the proportion of different iso-tRNA species is correlated with the square root of codon frequency. Quantitations of reticulocyte tRNA support such a parabolic relation. Our translation system model brings some light into the role of tRNA adaptation for optimizing translation efficiency, i.e. maximizing both speed and accuracy. Some consequences of the model are discussed.

Structural studies on RNA from Bombyx mori L. I. Nucleoside composition of enriched tRNA species from the posterior silkgland purified by coutercurrent distribution.

A large scale fractionation of tRNA from the posterior silkgland of the silkworm Bombyx mori L. by countercurrent distribution is described. One single 1,500 transfer distribution carried out with Phosphate buffer-Fromamide-Isopropanol (PFI) solvent system yields highly enriched isoaccepting species with increasing mobility order: tRNA1Gly, tRNA1-2Ala, tRNATyr, tRNA2Gly, tRNA1Ser and tRNA2Ser with 75%, 70%, 90%, 60%, 60%, and 90% purities respectively. Nucleosides fingerprint analysis of each iso-tRNA species confirms the anticodon structures previously suggested for tRNA2Ala (IGC), tRNA2bGly (U-CC) (U-CC) and tRNA2bSer (IGA). Twenty two minor nucleosides, three of them with unknown structure, have been detected. They are: m5C in tRNA1Gly, m1I in all tRNAAla species, polar A and U called X in tRNATyr, polar U derivative in tRNAGly2, mt6A in tRNASer1 and i6A tRNA2Ser. Both tRNASer species have m3C and ac7C. We do not detect Q, Y and thiol derivatives. The elution characteristics of silkgland tRNA species may be expressed in a semilogarithmic diagram where log K (K is the partition coefficient) is related to the base ratio A/Y) and the coding properties. The distribution pattern of silkgland tRNAs has been compared with that of Yeast and Rat liver tRNAs fractionated by countercurrent distribution with the PFI and PMB (Potassium phosphate buffer, 2-methoxy ethanol, 2-butoxy ethanol) solvent systems.

Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2.

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for the prediction of chemotherapeutic success in natural bovine infections with F. hepatica. Lactating cows (n = 16) from a herd naturally infected with F. hepatica were successively treated with nitroxynil (Dovenix, Specia) and with oxyclozanide (Zanil, ICI) 1 month later. Their f2-specific antibodies were significantly lower than those of a non-treated control group (n = 15) from the second month after the first treatment, and continued to decline thereafter to negative values 5-6 months post-treatment. In a second experiment, culled and fattened cows (n = 32) of unknown fasciolosis history were treated with closantel (Janssen Pharmaceutica). Three months after treatment, f2-specific antibodies of the serologically positive animals (n = 24) were reduced nine-fold. In contrast, in the control group (n = 28), the titers of f2-specific antibodies of the serologically positive animals (n = 21) were not modified significantly. The results show that the f2-HA test is useful for the prediction of chemotherapeutic success in bovine fascioliasis.

Decrease in cow-calf attachment after weaning.

Experiments were undertaken to measure attraction of suckler calves to their dams after separation for abrupt weaning. On days 0, 2, 7 and 20 after weaning in experiment 1 and on days 1, 9, 16, 24 and 35 after weaning in experiment 2, calves were observed in a pen where they could stay either near the dam or near another familiar cow or calf. On days 0, 2, 7, 20 and 35, calves and their dams were reunited for 2 min. Calves showed a preference for the dam to another cow up to day 24 and to a calf up to day 9 only. From day 20, cows rejected their calves at suckling. It is concluded that the calves remained attracted by their dams for at least three weeks after weaning while the cows already rejected them. In addition, attraction between calves increased after weaning.

Effect of cattle grazing a species-rich mountain pasture under different stocking rates on the dynamics of diet selection and sward structure.

Although stocking rate is a key management variable influencing the structure and composition of pastures, only few studies have simultaneously analysed the seasonal patterns of pasture use by cattle, and the adjustments the animals make to maintain intake of a high-quality diet over the grazing season. Therefore, over a 3-year study, we recorded diet selection, plot use and impact of heifers on sward structure and quality under three different stocking rates (0.6, 1.0 and 1.4 livestock units (LU) per ha) in a species-rich mountain pasture of central France. Measurements were made on three occasions between early June and the end of September each year. Overall, heifers selected for bites dominated by legumes or forbs, and against reproductive grass, whatever the stocking rate or season. Selection for tall mixed (P < 0.05), short mixed (P < 0.05) and short pure grass bites (P < 0.01) was more pronounced in plots grazed at the lowest stocking rate. Although heifers' selection for short patches decreased at the end of the season (P < 0.001), they continued to graze previously grazed areas, thus exhibiting a typical 'patch grazing' pattern, with the animals that grazed at the lowest stocking rate tending to better maintain their selection for short patches in September (treatment × period: P = 0.078). Neither diet quality nor individual animal performance were affected by the different stocking rate treatments despite high variability in the quantity and quality of herbage offered and differences in diet selection. However, at the 1.4 LU per ha stocking rate, the quantity of forage available per animal at the end of the season, 0.79 t dry matter (DM) per ha of green leaves with the median of sward height at 4.6 cm, approached levels limiting cattle's ability to compensate for the effects of increasing stocking rate. In plots grazed at 0.6 LU per ha, the total herbage biomass remained higher than 3 t DM per ha with more than 30% of plot area still covered by reproductive grass patches at the end of the grazing season, which in the medium term should affect the botanical composition of these pastures. Sward heterogeneity was high in plots grazed at 1.0 LU per ha, with sufficient herbage availability (1.1 t DM per ha of green leaves) to maintain animal performance, and more than 15% of plot area was kept at a reproductive stage at the end of the grazing season. Hence, it could represent the optimal balance to satisfy both livestock production and conservation management objectives.

Differential synthesis rates of tRNA species in the silk gland of Bombyx mori are required to promote tRNA adaptation to silk messages.

In studying the mechanism of tRNA adaptation to silk fibroin and sericin mRNA translation, we investigated the accumulation rates of tRNA species rapidly labeled in vivo in the posterior and middle silk glands of the silkworm Bombyx mori during the last larval instar. We found that the rates of synthesis of total and individual tRNA species labeled with [3H]uridine during the growth and the secretion phases and separated by electrophoresis on polyacrylamide gels, are specific for each tRNA species and each tissue. We noted continuous variations of the tRNA levels designed to reflect changes in the population of mRNAs. The tRNA adaptation to silk mRNAs is completed roughly at the end of the growth phase and at the beginning of the secretion phase (day four of the Vth instar). Its stabilization during the secretion phase occurs by a reversal of the relative rates of predominant tRNA species, mainly tRNAGly. The sum total of the synthetic rates for each tRNA species corroborates their known adaptive distribution during the secretion phase of silk proteins. In addition, when coupled to the similarity of their turnover rates, observed previously, the data support a transcriptional control of tRNA genes.

[Pathology and productivity of the lactating cow: interrelations based on the level of lactation]. / Pathologie et productivité de la vache laitière: interrelations à l'échelle de la lactation.

The effects of sanitary problems in dairy herds on milk production have been determined using 595 cases of health problems noted in Friesian and Montbeliarde cows in a long-term feeding trial, covering over 487 lactations. Relationships between each sanitary condition and lactation characteristics were established. On a short-term basis (5 weeks), the greater milk losses were first due to lameness at turnout (56 kg) and second to winter mastitis (24 kg); the effects of lameness lasted beyond the 5th week depending on when the problem was first detected. Over the whole lactation cycle, the highest milk losses resulted from recurrent lameness: 640 kg loss for cows presenting a lameness at least three times, versus 20 kg for those presenting only one lameness. The frequency of recurrent lameness was three times higher in Friesian than in Montbeliarde cows and four times higher with grass silage-based diets as compared with hay diets. Four main types of lactation cycles were characterized on the basis of the cows' level of production, their health status, their reproduction performances and their culling rate. Cumulative differences in any of those characteristics accounted for up to a 1 800 kg difference in milk production.

Differential usage of iso-accepting tRNASer species in silk glands of Bombyx mori.

The rapid development of the silk glands of Bombyx mori during the last larval instar shows two phases. During the first 4 days, in both the middle and posterior parts of the silk glands, the ribosomal machinery is assembled and the synthesis of housekeeping proteins starts. During the second phase (the last 4 days), the middle part of the gland synthesis approximately 45 mg of the silk protein sericin (31% serine) and the posterior part of the gland synthesizes approximately 130 mg of the silk protein fibroin (46% glycine, 29% alanine and 12% serine). Silk fibroin and sericin are detectable by the second day and represent 80 and 50% respectively of the total proteins produced at day 8 (refs 1--4). It is known that the tRNA population of the posterior part of the gland is quantitatively adapted to fibroin codon frequency during this period but little is known about the situation in the middle part except for the observation that it contains more tRNASer than does the posterior part. We show here that the two parts contain, and presumably use, different iso-accepting species of tRNASer, the middle part using tRNASer1, which recognizes AGU and AGC codons, and the posterior part using tRNASer2 which recognizes UCA. We also suggest that this differential adaptation of the tRNASer species is under transcriptional control as the two species are accumulated at different rates, but degraded at the same rate.

[Feeding, pathology and productivity of the dairy cow: interrelations during the course of lactation]. / Alimentation, pathologie et productivité de la vache laitière: interrelations à l'échelle de la carrière.

The influence of winter feeding (hay versus grass silage-based diets, supplemented with a high or low level of concentrate) and disease on the longevity of 136 Friesian or Montbeliarde dairy cows was studied in a long-term experiment spanning 6 consecutive years. Breed had no great effect on longevity. The longevity of cows given a silage diet with a low level of supplementation was lower than that of cows given any one of the other diets (2.5 lactation/cycle versus 3.2-3.5). Cows that developed frequent lameness during the 1st lactation had a shorter period of productivity (-1.1 lactation/cycle) than comparable healthy animals. Among the different pathological lactation profiles, only the profile "healthy lactation" recurs from one lactation to the next (41% of the cases). In particular, lameness (which is very recurrent during a single lactation) is not recurrent from one lactation to the next. The characteristics of the 1st lactation cycle (disease, milk production, reproduction) appeared to be determinant for the productive future of the animals. On average, milk production and live-weight increased by 352 and 24 kg respectively between the 1st and 2nd lactation and by 270 and 27 kg between the 2nd and 3rd lactation. Cows given a hay diet (with a low or high level of supplementation) or a silage diet with a high level of supplementation showed a greater increase in production between the 1st and 3rd lactation (+ 752 kg) than those given a silage diet with a low level of supplementation (+ 359 kg). Over 3 lactations, the cumulative effects of the type of winter feeding can become marked: differences in milk production can attain up to 2770 kg between a hay/high concentrate and a silage/low concentrate diet. These results question the validity of conclusions drawn from experiments conducted over 1 winter or 1 lactation cycle only.

[tRNA adaptation and the optimization of translation]. / Adaptation des tRNA et optimisation de la traduction.

The intracellular level of each tRNA species is adjusted to the codon frequency of the mRNA being decoded. This was first observed in such highly differentiated cells as the silk gland of Bombyx mori, which produces fibroin and sericin, and the rabbit reticulocyte. tRNA adaptation also occurs in other cell types from E. coli to mammalian cells. Regardless of the mechanism regulating tRNA biosynthesis, we believe that tRNA adaptation is the basic step optimizing chain elongation at the ribosomal level. We propose the system of trial and error as a working model for the ribosome. This model clarifies the correlations between iso-accepting tRNA levels and codon frequencies, as well as the effect of tRNA pool balance on mean elongation rate and non-uniform individual elongation rate (depending on whether codons are rare or abundant) for fibroin mRNA translated in a reticulocyte cell-free system.

[Characterization of the pathology of lactating cows based on the level of lactation. Principal factors in the variation and typing of pathologic profiles of lactation]. / Caractérisation de la pathologie de la vache laitière à l'échelle de la lactation. Principaux facteurs de variation et typologie des profils pathologiques de lactation.

The health disturbances investigated were observed during a long-term trial (six years) conducted at an experimental station located at 1,100 m elevation. The study dealt with 487 lactations involving 190 cows of the Montbéliarde and French Friesian breeds, which produced on average 4,200 kg milk per lactation. The disturbances concerned 59% of monitored lactations, with a mean incidence of 2.1 disturbances per lactation. Lameness and mastitis accounted respectively for 52 and 24% of the clinical affections. Pathology was significantly influenced by breed, basic diet (hay or grass silage), concentrate quantities, lactation rank and year. The authors describe a method permitting an independent analysis of the effects of lactation stage and of season on mastitis and lameness frequency, by limiting the biases due to grouping of calvings and to culling. The study of lactations affected by several pathological disturbances shows that the different types of affections recorded are mutually independent but that successive occurrences of the same affection are not. On the basis of these results, the authors have proposed to globally characterize the "pathological profiles" of lactations.