Genetic relationship between o6 and pro-1 mutants in maize

Summary This paper reports that the opaque-6 (o6) mutation of maize, which causes seedling lethality and interferes in the endosperm with the synthesis of zeins and b-32 protein, is a proline requiring mutant functionally allelic to proline-1 (pro-1). Furthermore, immunological studies on the b-32 content of ten independently originated o6 and pro-1 alleles demonstrated that four alleles contain an apparently normal b-32 protein while the others are either devoid of it or contain trace amounts of cross-reacting proteins of lower molecular weight.     

Southern elephant seal,Mirounga leonina: composition of milk during lactation

An analysis of milk constituents during various stages of lactation in the southern elephant seal Mirounga leonina was carried out. Forty-six milk samples were taken from 30 females throughout lactation during 1985, 1987, 1990 and 1991 on Stranger Point, King George Island, South Shetland Islands. Total nitrogen (TN), non-protein nitrogen (NPN), sugar, fat, ash and water were measured, and from some of these data true protein and energy content were calculated. The results showed a high degree of variation in water and fat concentrations among samples at different stages of lactation. During the first 20 days the fat content of milk increased from about 12 to approximately 52%, while water content fell from 70 to 33%. The composition of milk changes rapidly during the first days post-partum. Protein, minerals and sugar appear to remain stable after the fourth day of lactation. Milk samples contain significant levels of sugars; thin layer chromatography indicates the presence of lactose and glucose together with other unidentified components. There is evidence of a striking change in composition of the milk in the later part of lactation; the progressive increase in the fat:water ratio is abruptly reversed just prior to weaning.     

Amino acid composition of zein molecular components

Zein extracted from maize endosperm has been fractionated into four polypeptide chains, having the following MWs 23 000, 21 000, 13 500 and 9600. By amino acid analysis the two smaller MW chains (representing 30% of total zeins) have been found to be zein-type molecules. These two chains are thought to be responsible for zein granule formation via -S-S- bridges. Zein is also highly heterogeneous in charge, and is resolved into at least 15 components, with pI's in the pH range 5–9. As demonstrated by amino acid analysis, part of this heterogeneity is due to spot mutations in some of the genes responsible for zein synthesis.     

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000     

Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize

Summary The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.     

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De^*-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.     

Lithium chloride-induced primary cilia recovery enhances biosynthetic response of chondrocytes to mechanical stimulation

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0–50 mM LiCl for 24 h, with different pre-culture durations (0 to 4 days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50 mM LiCl compared to other concentrations (P?<?0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50 mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P?<?0.1) and proteoglycan (1.5-fold, P?<?0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.

     

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O₂ and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.