Analysis of productivity in lysis-deficient lambda expression systems

The integrated state of lambda in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on lambda expression systems(21,22) have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of lambda systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential lambda protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector lambdaZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in lambda systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of lambda vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. (c) 1992 John Wiley & Sons, Inc.     

A comparison of neural networks and partial least squares for deconvoluting fluorescence spectra

This article compares backpropagation neural networks (BNN) with partial least squares (PLS) techniques in terms of their ability to deconvolute fluorescence spectra. Both actual experimental and simulated spectral data are studied for 2 binary systems. These systems consist of mixtures of tryptophan and tyrosine, and NADH and tryptophan over a total concentration range of 10(-7) to 10(-4) M. It is shown that BNN is superior to PLS for both systems.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

Protein analysis during the ontogeny of normal and male sterile stamenless-2 mutant stamens of tomato (Lycopersicon esculentum Mill.)

The levels and synthesis of proteins during the ontogeny of normal and male sterile stamenless-2 (sl-2/sl-2) mutant stamens of tomato (Lycopersicon esculentum) were examined. The mutant stamens contained low levels of soluble protein which were related to reduction in protein synthesis. The mutant stamens, however, possessed many polypeptides similar to the normal and synthesized a 53-kd polypeptide at stages when there are abnormalities in tapetum development. The mutant stamens also possessed a 23-kd and some low molecular weight polypeptides that were considered as degradative proteins. Normal stamens exhibited the synthesis of many polypeptides not found in the mutant, from microspore mother cell to the preanthesis stages. In addition, at the time of pollen maturation there was a greater synthesis of several polypeptides, particularly those of 42 and 37 kd. Although the causative mechanisms of male sterility in the sl-2/sl-2 mutant are not known, the synthesis, and the lack, of specific polypeptides reported here appears to be associated with pollen degeneration.This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to V.K.S.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

A method for subgrouping the S-type of CMS forms in maize

Summary Simple correlations were calculated between nine different cms sources (cms-S, -R, -ML, -L, -CA, -EK, -C, -Rb, -T) on the basis of the weighted restoring reactions of 41 inbred lines. The Principal Component Analysis was applied to a 9 × 9 matrix which clearly grouped cytoplasms according to their similarities. The Principal Component I included S, R, ML, L, CA and EK cytoplasms; the Principal Component II contained C and Rb cytoplasms while T-cytoplasms was placed in Principal Component III. This corresponds to the main groupings indicated in the literature (Beckett 1971). However, after varimax rotation of the Principal Components, the S main group, including the 6 tested cytoplasms, fell into 3 subgroups: I.: S, R, ML; II.: L, CA; III.: EK.These data indicate that the Principal Component Analysis can be used to select a limited number of cms sources from the S group, representing the variability of the cytoplasmic gene pool of that group.     

Differences between genetic and physical centromere distances in the case of two genes for male sterility in barley

Summary Linkage studies with thirty translocations (one of the two chromosomes involved being number 4) in relation to msg24 (chromosome 4) and thirteen translocations (one of the two chromosomes involved being number 6) in relation to msg6 (chromosome 6) show without exception close linkage for all combinations tested. The results indicate that both genes are located genetically in or close to the centromere regions of their chromosomes.Cytological analysis of two BTT stocks (balanced tertiary trisomics) ascertained the respective chromosome arms (both msg24 and msg6 on the short arms) and revealed marked differences between genetic and physical centromere distances. The reason is obviously the high content of centromeric heterochromatin occupying both the chromosome arms involved.     

Amino acids in anthers of Milo and in cytoplasmic genetic male sterile sorghums (Sorghum bicolor L. Moench) of Indian origin

Summary Amino acid composition of proteins from anthers of milo and Indian origin male steriles were determined. Comparison of amino acid between A and B lines showed lower contents of histidine, threonine, glutamic acid, glycine, leucine and phenylalanine and higher contents of alanine, serine, proline and tyrosine in line A compared to line B. Alanine content in anthers of A lines was more than two fold higher than that in the anthers from B lines. Marked differences in amino acid composition of anthers of A and B lines are suggestive of their involvement in male sterility. Cytoplasmic male steriles of Indian origin M35-1A and M31-2A showed greater similarity but differed from milo, VZM2A and B.     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Model for genetic control of Heliothis virescens

Summary A model was developed which corrects and extends an earlier one proposed for the control of the tobacco budworm, Heliothis virescens (F.), through hybrid male sterility. Population suppression is effected through the release into natural populations of the backcross progeny of a hybrid between H. virescens and a related species. Thereafter, the system perpetuates itself in nature through continual backcrossing of the fertile backcross females to native H. virescens males. When the proportion of backcross hybrid females in the total population is large enough to draw off the insemination potential of the native males, the native females fail to replace themselves. The present model demonstrated that the ratio of released backcross hybrids to natural H. virescens remains constant in a closed population. Furthermore it was shown that the release ratio necessary to achieve extinction of a closed population is related to the number of females that a male can inseminate and to the population growth rate. Release ratios required to slow natural population growth and to lessen the impact damage of releases on crop plants were also examined. Effects of selection against the backcross females on the predictions of the model were explored.