Three sucrose transporter genes are expressed in the developing grain of hexaploid wheat

A family of three cDNAs, designated TaSUT1A, 1B and 1D, encoding sucrose transporter (SUT) proteins was isolated from a hexaploid wheat (Triticum aestivum) endosperm library. The cDNA sequences are 96% identical but are distinguishable from one another by virtue of a size polymorphism in the 3-untranslated region (UTR). The predicted amino acid sequences are 98% identical and are highly similar to the sucrose transporters from rice, maize and barley. A gene for TaSUT1 was isolated from genomic libraries of Aegilops tauschii (the donor of the D genome of wheat) and the coding sequence found to be identical to that of TaSUT1D cDNA. There is only one copy of each TaSUT1 gene in hexaploid wheat and it is located on chromosome 4. Genomic Southern analysis and PCR analysis across the 3 polymorphic region of hexaploid, tetraploid and progenitor diploid wheat DNAs established that the TaSUT1A gene was present in the putative A-genome progenitor, T. monococcum, and that the TaSUT1B gene was present in the putative B-genome progenitor, T. searsii. All three TaSUT1 genes are expressed at high levels in filling grain, showing a good correlation with the developmental time course of growth. This reinforces the view that in cereals a major role of SUT1 is in the post-phloem sugar transport pathway associated with seed filling.     

Tumor promoter-inducible genes are differentially expressed in the developing mouse.

TIS genes are rapidly and transiently induced by tetradecanoyl phorbol acetate in 3T3 cells. We analyzed the developmental appearance of a number of the TIS genes to determine whether, in a normal physiological context, these genes have common or distinct mechanisms of regulation. Each TIS gene has a distinct tissue specificity and/or developmental profile.     

Multiple genes encoding zinc finger domains are expressed in human T cells

Proteins containing zinc finger domains have been implicated in developmental control of gene expression in Drosophila, Xenopus, mouse, and humans. Multiple cDNAs encoding zinc (II) finger structures were isolated from human cell lines of T-cell origin to explore whether zinc finger genes participate in the differentiation of human hematopoietic cells. Initial restriction analysis, genomic Southern blotting, and partial sequence comparisons revealed at least 30 nonoverlapping cDNAs designated cKox(1-30) encoding zinc finger motifs. Analysis of cKox1 demonstrated that Kox1 is a single-copy gene that is expressed in a variety of hematopoietic and nonhaematopoietic cell lines. cKox1 encodes 11 zinc fingers that were shown to bind zinc when expressed as a beta-gal-Kox1 fusion protein. Further analysis of the predicted amino acid sequence revealed a heptad repeat of leucines NH2-terminal to the finger region, which suggests a potential domain for homo- or heterodimer protein formation. On the basis of screening results it was estimated that approximately 70 zinc finger genes are expressed in human T cells. Zinc finger motifs are probably present in a large family of proteins with quite diverse and distinct functions. However, comparisons of individual finger regions in cKox1 with finger regions of cKox2 to cKox30 showed that some zinc fingers are highly conserved in their putative alpha-helical DNA binding region, supporting the notion of a zinc finger-specific DNA recognition code.     

Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

Proteolytic enzymes in wheat leaves were studied using zymographic detection of enzyme activities on one-way (1D) SDS-polyacrylamide gels and two-dimensional (2D) ones, on which protein samples were isoelectrofocused prior to PAGE separation. Gelatin of concentration 0.1 %, copolymerized into SDS-PAGE gels, digested by active proteinases enabled detection of those enzymes. On 1D gels, seven bands were seen and assigned to particular families through the use of specific inhibitors. Metalloproteinases inhibited by 20 mM EDTA were detected as 150 kDa band; aspartic proteinases were assigned to 115–118 kDa double band by using 25 mM pepstatin; 10 mM phenylmethylsulfonyl fluoride used for detection of serine proteinases pointed to band of 70 kDa and finally due to 10 μM E-64 inhibitor, cysteine proteinases of 37 and 40 kDa were detected. On 2D gels, additional separation according to protein isoelectric points enabled detection of proteinase isoforms. In the range of 4.5–6 pI, six metalloproteinases as well as ten aspartic proteinases were visible, ten serine- isoforms of pI 4.5–6.8 and four cysteine proteinases of 4.5–5.0 pI were found. Presented results were detected as reproducible results observed at least in four independent biological replications.     

Structure of genes encoding steroidogenic enzymes

Synthesis of adrenal steroid hormones from cholesterol entails the actions of only five enzymes, four of which are specific forms of cytochrome P450. These cytochrome P450 enzymes have all been isolated and their activities reconstituted in vitro, showing that each enzyme catalyses multiple steroidal conversions. Genes or complementary DNAs have been cloned for human P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase). The sequences for microsomal P450c17 and P450c21 are much more closely related to one another than either is to the sequence for mitochondrial P450scc. Each of these P450 enzymes is encoded by a single human gene; the gene for P450scc lies on chromosome 15, that for P450c17 lies on chromosome 10, and that for P450c21 lies on chromosome 6. The human, mouse and bovine genomes each have two P450c21 genes. While only one of these is active in mouse and man, both genes may be active in cattle. A wide variety of lesions in the human P450c21(B) gene causes congenital adrenal hyperplasia, a common genetic disorder.     

Environmentally-induced changes in protein composition in developing grains of wheat are related to changes in total protein content

Nitrogen (N) nutrition, post-anthesis temperature and drought-induced changes in the kinetics of accumulation of dry mass, total grain N and protein fractions (albumins-globulins, amphiphils, gliadins, and glutenins) contents were examined for winter wheat (Triticum aestivum L.). Crops were grown in controlled environment tunnels in 1994 and 1998. In 1994, five post-anthesis temperatures averaging from 15-25 degrees C were applied during grain-filling. In 1998 two post-anthesis temperatures averaging 13 degrees C and 20 degrees C were applied and factorized with two post-anthesis water regimes. In 1994 crops also were grown in the field, where different application rates and timing of N nutrition were tested. When expressed in thermal time, the kinetics of accumulation of the protein fractions were not significantly affected by post-anthesis temperature or drought; whereas N nutrition significantly increased the rate and duration of accumulation of storage proteins. Albumin-globulin proteins accumulated during the early stage of grain development. The rate of accumulation of that fraction decreased significantly at c. 250 degrees Cd after anthesis, when the storage proteins (gliadins and glutenins) started to accumulate significantly. Single allometric relationships for the different environmental conditions exist between the quantity of each protein fraction and the total quantity of N per grain. From these results it was concluded that (1) the process of N partitioning is neither significantly affected by post-anthesis temperature or drought nor by the rate and timing of N nutrition and (2) at maturity, variations in protein fraction composition are mainly because of differences in the total quantity of N accumulated during grain-filling.     

Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth

An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1, 4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly.     

Multiple non-allelic genes encoding pancreatic alpha-amylase of mouse are expressed in a strain-specific fashion.

The number of active Amy-2 genes has been estimated in strain CE/J mice which produce four distinct electrophoretic forms of alpha-amylase in their pancreas. cDNA cloning and DNA sequence analysis discloses five distinct mRNA sequences which differ by approximately 1% of their nucleotides. Two of these mRNAs specify the same protein. Changes in the nucleotide sequences result in amino acid replacements that alter the net charges of the deduced proteins. This has allowed a tentative assignment of individual mRNAs to isozymes detected by electrophoresis. Quantitative Southern blot hybridization using a DNA probe specific for the first exon of Amy-2 reveals the presence of greater than 10 Amy-2 related sequences per haploid CE/J genome. Models which could account for the mouse strain-specific differences with respect to the number of pancreatic alpha-amylase isozymes and their variable but genetically determined quantitative ratios are discussed.     

Fluoride-induced alteration of carbon and nitrogen metabolism in developing wheat grains

Effect of fluoride (10 and 50 mM) on the activities of sucrose metabolizing enzymes, alkaline inorganic pyrophosphatase, and transaminases in relation to the accumulation of free sugars, starch, and soluble protein was studied in detached ears of wheat cultured in a liquid medium. Culturing for 5 d in the presence of fluoride reduced the amount of grain starch whereas contents of total free sugars, particularly sucrose, and soluble protein increased. Fluoride inhibited the activities of soluble acid and neutral invertases, as well as sucrose synthase acting in the cleavage direction. Uptake of uniformly labelled ¹⁴C-sucrose or fructose was also drastically reduced by fluoride. Glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities also increased with fluoride addition in correspondence with an increase in soluble protein. Apparently, the wheat grain responds to fluoride-mediated disruption of carbon metabolism by a compensatory effect on nitrogen metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vacuoles are not the sole compartments of proteolytic enzymes in yeast

Localization in vacuoles, the lysosome-like organelle of yeast, was checked for several newly detected proteolytic enzymes. While aminopeptidase Co and carboxypeptidase S were found in vacuoles, proteinase D and proteinase E as well as a variety of other proteolytic activities detectable with the aid of chromogenic peptide substrates do not reside in this cell compartment.