Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment

It has been proposed that transgenic plants of cereals can be generated by inoculating florets with Agrobacterium at or near anthesis. This procedure is shown to lead to the production of embryos of wheat and barley with enhanced resistance to antibiotic selection. It has also been possible to recover plants of wheat, barley and maize that gave positive hybridization signals with probes produced from within the T-DNA of the Agrobacterium vector. However, no evidence was found for transmission of the bands detected by hybridization in the progeny of the putative transgenic plants nor could enzyme activity associated with the resistance genes be found in plant extracts. Furthermore, undigested genomic DNA from the plants that were positive when probed with the T-DNA, showed hybridization to bands smaller than the genomic DNA. It is suggested that the apparent transformation is an artifact of the procedure and does not reflect transformation of the plant nuclear genome.     

Molecular mechanics studies of dermorphin

Molecular mechanical simulations have been carried out on dermorphin. Presence of D-Ala2 at the N-terminus and L-Pro6 residue at the C-terminus indicated the probability of beta-turns. From the stereochemical considerations, three types- II', III' and V' - for the beta-turn at the N-terminus of the peptide and two types-I and III- for the C-terminus side of the peptide are possible. In our molecular mechanics calculations, we considered six folded and one extended conformations for dermorphin to asses the relative stabilities. Three of the six folded conformations are lower in energy and have the following general feature-similar in energy, three hydrogen bonds, semirigid beta-sheet segment and favorable Tyr1-Tyr5 interaction. The presence of beta-sheet structure might play a role in mu-receptor selective interaction of dermorphin.     

Construction and expression of synthetic DNA fragments coding for polypeptides with elevated levels of essential amino acids

Summary Polypeptides, with elevated levels of essential amino acids, could be useful as partial protein supplements to food and feeds. To obtain DNA fragments coding for these polymers, oligonucleotides were constructed by random synthesis of a mixture of appropriate codon pairs and inserted into a bacterial plasmid in E. coli. Two of the isolated fragments were subjected to DNA sequence analysis and theoretically code for polypeptides containing up to 23% lysine, 12% tryptophan, 12% methionine, 6% isoleucine, and 6% threonine. These five amino acids make up 60% of the total amino acid content of the peptide, compared with 25% for the same amino acids in lactalbumin, a milk protein considered to be high in essential amino acids. These fragments, when fused to an active bacterial promoter, which directs the synthesis of chloramphenicol acetyl transferase (CAT), cause bacteria, harboring these modified genes, to take up more lysine as compared to control cells and produce commensurately larger CAT polypeptides. This method of gene synthesis may permit production of polypeptides with a specified amino acid composition to supplement specific diets low in the essential amino acids.     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

Flexibility of 3',5' deoxyribonucleoside diphosphates

In 3',5' deoxyribonucleoside diphosphates, in addition to the nature of the base and the sugar puckering, there are six single bond rotations. However, from the analysis of crystal structure data on the constituents of nucleic acids, only three rotational angles, that are about glycosyl bond, about C4'-C5' and about C3'-O3' bonds, are flexible. For a given sugar puckering and a base, potential energy calculations using non-bonded, electrostatic and torsional functions were carried out by varying the three torsion angles. The energies are represented as isopotential energy surfaces. Since the availability of the real-time color graphics, it is possible to analyse these isopotential energy surfaces. The calculations were carried out for C3' exo and C3' endo puckerings for deoxyribose and also for four bases. These calculations throw more light not only on the allowed regions for the three rotational angles but also on the relationships among them. The dependence of base and the puckering of the sugar on these rotational angles and thereby the flexibility of the 3',5' deoxyribonucleoside diphosphates is discussed. From our calculations, it is now possible to follow minimum energy path for interconversion among various conformers.     

Infection of Locusta migratoria with entomopoxviruses from Arphia conspersa and Melanoplus sanguinipes grasshoppers

Entomopoxvirus (EPV) occlusion bodies isolated from Arphia conspersa and Melanoplus sanguinipes grasshoppers were fed to 3rd and 4th instar Locusta migratoria nymphs. Locus mortality induced by A. conspersa EPV was first detected 18 days after addition of virus to the diet, and reached a level of approximately 68% of the colony population by 60 days after virus inoculation. In a similar population of L. migratoria nymphs, mortality induced by M. sanguinipes virus reached 90% 60 days after virus inoculation. Entomopoxvirus was isolated from M. sanguinipes EPV infected locust nymphs and the viral DNA was cleaved with several restriction endonucleases. The DNA fragment patterns obtained after agarose gel electrophoresis were compared with the fragment patterns from the original sample of M. sanguinipes EPV DNA cleaved with the same restriction endonucleases. No differences in the cleavage patterns were detected between the two virus DNA samples. Virus structural proteins of M. sanguinipes EPV purified from infected locust nymphs were compared by polyacrylamide gel electrophoresis with virus proteins isolated from the original sample of M. sanguinipes EPV. A total of six different virus protein bands were detected between the two poxvirus preparations.     

Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication

The titer of Amsacta entomopoxvirus (EPV) protein detected in murine L-929 cells by enzyme-linked immunosorbent assay (ELISA) decreased to within preimmune serum levels by 24 hr after inoculation of the virus which indicates that Amsacta EPV structural protein biosynthesis does not occur in the vertebrate cell line. A viral-induced protein of approximately 100,000 M_r was detected by [³⁵S]methionine incorporation 4 hr after inoculation of Tn-368 cells with Amsacta EPV. Biosynthesis of protein which reacted with vaccina antiserum was detected in Estigmene acrea (BTI-EAA) cells by ELISA 10 hr after inoculation with 10 PFU of virus per cell. The amount of putative vaccinia structural protein detected in BTI-EAA cells increased approximately twofold by 70 hr after virus inoculation. No increase in vaccinia structural protein biosynthesis was detected in BTI-EAA cells inoculated with vaccinia virus previously inactivated by heat and UV light.     

Mutations conferring quantitative and qualitative increases in beta-galactosidase activity in Escherichia coli

Summary Sodium lactobionate is not utilized as a carbon source byEscherichia coli because it is only poorly bound and hydrolyzed by -galactosidase and it does not induce the formation of the enzyme. However, treatment with N-methyl-N-nitro-N-nitrosoguanidine produced 32 independent mutants able to grow on lactobionate. Most of the mutants formed -galactosidase constitutively, 29 of them having mutations in the regulatory gene and one possibly in the operator. In addition, the mutants possessed quantitatively—or qualitatively—altered -galactosidase. In 28 mutants the -galactosidase activity was 1.5 to 4.5 times that of the wild-type. The enzymes of these mutants were unaltered in thermostability and substrate binding. One enzyme that was titrated immunologically possessed a molecular activity indentical with the wild-type enzyme. These mutants appear to contain extra copies of the gene for -galactosidase. The spontaneous mutation rate to constitutivity was 6.3x10^-3 and to the formation of apparently extra genes, 9.2x10^-3.The -galactosidases of three mutants were qualitatively changed as judged from their increased thermosensitivity, altered substrate-binding constants and greatly increased ability to hydrolyze lactose and lactobionate. Affinity for 0-nitrophenyl--galactoside and galactose was increased by the mutations while that for lactose was decreased; maximum velocities for the hydrolysis of 0-nitrophenyl--galactoside were also decreased. Relative to their rates of hydrolysis of 0-nitrophenyl--galactoside, these altered enzymes hydrolyzed lactose at 6 to 8 times, and lactobionate up to 23 times, the rate given by the normal enzyme. The mutations appear to increase the hydrophobic nature of the enzyme near the aglycon binding site and facilitate the hydrolysis of more hydrophilic galactosides. The lactobionic acid positive character could be transferred to other bacteria by sexual conjugation when the enzyme changes were qualitative, but not when they were quantitative.     

Thermal responses of mutant enzymes and temperature limits to growth

Summary The half-lives of fifty-two mutant forms of -galactosidase, in which serine had been substituted for the original amino-acid, were measured at 57°C. Sixty percent of the mutant enzymes had less than half the stability of the normal enzyme at high temperature but none had greater sensitivity to low temperature. A sample of these thermolabile enzymes showed unaltered activation energy. Enzymes with altered substrate affinity generally had reduced apparent heats of activation, decreased heats and entropies of association, and were thus relatively less sensitive to temperature change.The intracellular behaviour of thermolabile enzymes was examined by showing that substrate hydrolysis is the limiting step in lactose utilization, that the enzyme-substrate complex is more stable than the free enzyme and that binding of the enzyme to structures within the cell increases stability and lowers affinity. Amongst enzymes having half-lives less than about seven minutes at 47.75°C, there is a close relationship between enzyme lability and diminished growth rate on lactose at 44°C. The effect of mutation on the thermal behaviour of multi-enzyme systems and the relation of enzyme thermolability to temperature-induced growth deficiencies and temperature-dependent heterosis are discussed.