Conservation and change in structural and 5' flanking sequences of esterase 6 in sibling Drosophila species.

Esterase 6 (Est-6/EST6) is the major beta-carboxylesterase in D. melanogaster and its siblings D. simulans and D. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least within D. melanogaster and D. simulans. Here we present the nucleotide sequences of an Est-6 allele and its flanking regions from each of D. simulans and D. mauritiana and compare them with the published D. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher for Est-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism within D. melanogaster and D. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5' of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (-1 to -158bp and -219 to -334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa, lucerne) and Onobrychis viciifolia Scop. (sainfoin).

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the "recipient" parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33-78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.

Extended target-site specificity for a hammerhead ribozyme.

In vitro mutagenesis has been used to systematically mutate the GUC target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a GUG target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discernable cleavage.

Inheritance of supernodulation in soybean and estimation of the genetically effective cell number.

Provided the nature of inheritance is known, the frequency of homozygous mutant plants in individual M2 families (derived from M1 seed) can be used to estimate the genetically effective cell number (GECN). Segregation ratios in M3 families derived from M2 wild-type plants indicated that the supernodulation characters nts382, nts1007 and nts183 are inherited as Mendelian recessives. The nature of inheritance was also known or confirmed to be recessive by crossing the wild type to these and several other mutants derived from the same population of M2 families. Subsequently, using the frequency of mutant plants in individual M2 families, the GECN for soybean was calculated to be approximately two.

Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors.

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.