The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS), which is normally subject to feedback inhibition by tryptophan. Three independent frp5 mutants defective in the Arabidopsis fbaliana AS a subunit structural gene ASA7 were identified by selection for resistance to the herbicidal compound 6methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspartate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration causing 50% inhibition of AS activity in the frp5 mutant increased nearly 3-fold, the apparent K,,, for chorismate decreased by approximately 50%, and the apparent V,,, increased 60%. As a consequence of altered AS kinetic properties, the frp5 mutants accumulated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in frp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. fbaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryptophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.The enzyme AS (EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate) pathway toward the biosynthesis of Trp. In addition to its primary role in providing an amino acid for protein synthesis, the Trp biosynthetic pathway of higher plants also produces precursors for synthesis of a variety of important secondary metabolites, including the phytohormone IAA (Normanly et al., 1995), antimicrobial phytoalexins (Tsuji et al., 1993, and other indolic molecules that influence plant-microbe and plant-animal interactions. As a branchpoint enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis (see Fig. 1 for a schematic of the pathway and tvp mutant designations).

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Some reports indicate that sperm from different males differ in capacitation time, and other reports suggest that freezing sperm may affect their capacitation time. These two variables were specifically studied in rabbits in a fertility trial with 96 does inseminated with approximately 1.6 million motile fresh or frozen sperm from three different bucks at 15, 10, 5, and 0 h before expected ovulation. Fresh semen averaged 84% live (unstained) sperm and 88% had normal acrosomes; corresponding values for frozen sperm were 44% and 54%. On the basis of does that became pregnant, average litter size with fresh semen was 5.5 and with frozen semen was 4.8 (p greater than 0.05), but overall, does bred with frozen semen produced fewer young (p less than 0.05). On the basis of total does and total semen, average litter size from insemination at 15, 10, 5, and 0 h was 2.8, 4.2, 3.8, and 1.7, and average litter size for the three bucks was 4.0, 1.8, and 3.6. There was no interaction of type of semen (fresh or frozen) with the other variables in the model (p greater than 0.05). Bucks and time of insemination affected both the proportion of does that were pregnant and litter size (p less than 0.01). A major interaction between buck and time of insemination (p less than 0.01) was due apparently to both differential sperm survival and probable capacitation time among bucks. This major interaction should be considered in designing in vitro and in vivo fertility studies, and for selecting males for use in artificial insemination.

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One-cell or two-cell rabbit embryos were cultured in protein-free media with various NaCl concentrations and osmolarity to determine relative sensitivity of embryos to changes in media composition. Embryos from replicates of donor rabbits were distributed randomly across treatments and cultured at 39 degrees C. Zygotes were cultured in Expts 1, 2A and B, and 3, and two-cell embryos were cultured in Expts 4A and 4B. In Expt 1, blastocyst formation and number of cells were highest (P < 0.05) in the control medium with 93 mmol NaCl l-1 (270 mosmols) compared with media containing 63, and 116 mmol NaCl l-1 (220 and 316 mosmols). In Expt 2, embryos were cultured in media with 70 or 93 mmol NaCl l-1, varying in osmolarity from 250 to 320 mosmols by adding sorbitol. In media with 70 mmol NaCl l-1 and osmolarities of 250, 280 and 300 mosmols, there were 41, 56 and 50% expanded blastocysts, respectively (P < 0.05). With 93 mmol NaCl l-1 and osmolarities of 270, 293 and 320 mosmols, embryos developed into 37.53 and 27% expanded blastocysts, respectively, (P < 0.05). In Expts 3A and 3B and 4A and 4B, the osmolarity of the medium was maintained at 270 mosmols by adding sorbito to media containing 40 or 60 mmol NaCl l-1, and other components were reduced in media containing 100 and 116 mmol NaCl l-1 to compensate for the higher NaCl. Zygote development into blastocysts was greatly suppressed (P < 0.05) in media with 40, 60, 100 and 116 mm NaCl l-1, compared with the control (93 mmol NaCl l-1), whereas development of two-cell embryos into blastocysts was much less affected. These results appear to reflect a direct sodium chloride as well as osmolarity effect on embryo development; and zygotes are much more sensitive to these effects than are two-cell embryos.