Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers

The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being α-amylase. The mRNA for α-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.     

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone

The effect of gibberellic acid and Ca²⁺ on the accumulation of α-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) α-amylases. There is no significant hybridization between the two α-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI α-amylases. It is shown that although the synthesis of the high pI α-amylase proteins depends on the presence of Ca²⁺ in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca²⁺. The accumulation of low pI α-amylase mRNA is also not affected by the presence or absence of Ca²⁺ in the incubation medium. These results establish gibberellic acid, not Ca²⁺, as the principal regulator of α-amylase mRNA accumulation in barley aleurone, while Ca²⁺ controls high pI α-amylase synthesis at a later step in the biosynthetic pathway.     

Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells.

We have determined the nucleotide sequences of Amy32b, a type A alpha-amylase gene, and of the gene for aleurain, a thiol protease closely related to mammalian cathepsin H. Both are expressed in barley aleurone cells under control of the plant hormones gibberellic acid and abscisic acid, but only aleurain is expressed at high levels in other barley tissues. Sequence analysis indicates that the 5' end of the aleurain gene, comprising 3 exons and 2 introns, may have become associated with the remainder of the gene, encoding the protease domain of the protein, by some sort of recombination event. This 5' domain of the gene is very G + C-rich and is flanked by inverted repetitive sequences. We found two different groups of homologous sequence elements. The first group consists of four blocks of sequences conserved in the same spatial arrangement in both genes; these are arranged at similar intervals upstream from the Amy32b TATA box and from a TATA box present in intron 3 of aleurain, outside of the 5' domain and upstream from the protease domain. A part of two of these conserved sequences is similar to the core sequence of certain enhancer elements characterized from mammalian cells. The second group of homologous elements is present in the upstream region of both genes. We speculate that these conserved sets of sequences may have some role in either the tissue specificity of expression of the genes or in some part of the hormonal regulation imposed on them.     

Detection of bioluminescence from individual bacterial cells: a comparison of two different low-light imaging systems

Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.     

Rat follistatin: ontogeny of steady-state mRNA levels in different tissues predicts organ-specific functions.

Follistatin (FS), a monomeric glycoprotein which specifically binds activin, is expressed in many tissues. This study investigated 1) the ontogeny of the steady-state FS mRNA levels in different extragonadal tissues and 2) whether the ratio of the differential splicing products, FS 344 or its carboxy-truncated form FS 317, is changed during postnatal development. Whereas the levels of FS mRNA 344 in the kidney showed a profound increase from the day of birth to adulthood, the levels in the muscle peaked during the infantile period and then declined. Brain cortex, heart and thymus also showed tissue specific expression in the steady-state mRNA level of FS during postnatal development. None of the tissues showed a measurable change in the ratio of the mRNA for FS 344 and FS 317. The FS mRNA 344 levels in male and female kidney were not different. It is concluded that the ontogeny of steady state FS mRNA varies in a tissue specific manner during postnatal development of the rat and may be involved in modulating the outcome of activin.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

Differential expression of the maize catalase genes during kernel development: the role of steady-state mRNA levels

In maize three isozymic forms of catalase, CAT-1, CAT-2, and CAT-3 are encoded by three distinct and unliked structural genes (Cat1, Cat2 and Cat3). Catalase activity profiles and zymogram analysis were used to examine the spatial and temporal expression of the three genes during kernel maturation. Three developmental stages of catalase expression were observed in the growing kernel. During stage 1 (6-12 days after pollination), both Cat1 and Cat3 were expressed; during stage 2 (15-18 days after pollination) only Cat1 expression was observed; and during stage 3 (21-30 days after pollination), Cat1 and Cat2 were expressed. The major constituent tissues of the kernel were examined to determine their contribution to total kernel catalase expression. Each of the tissues was found to have a unique pattern of catalase gene expression. RNA blot analysis, using catalase gene-specific nucleic acid probes, suggests that the differential expression of the three catalase genes observed in the kernel is regulated by controlling the distribution of steady-state mRNA species for the three genes.     

The mouse alpha-amylase multigene family. Sequence organization of members expressed in the pancreas, salivary gland and liver

The DNAs that specify the α-amylase messenger RNAs found in the pancreas, salivary gland and liver of mouse strain A have been isolated by molecular cloning in phage λ. Amylase clones were studied by mRNA/DNA hybrid analysis in the electron microscope, restriction endonuclease site mapping and DNA sequencing. The Amy-2^a gene, which specifies pancreatic α-amylase mRNA, measures 10·1 kb from cap to polyadenylation site and is interrupted by at least 9 intervening sequences. Amy-1^a, which specifies both salivary gland and liver α-amylase mRNAs contains at least 10 introns. The distance between the cap and polyadenylation sites used in the salivary gland and the liver measures 22·9 kb and 20 kb, respectively. Introns are located at very similar, if not identical, positions within comparable regions of Amy-1^a and Amy-2^a. The first intron of Amy-1^a, which interrupts sequences specifying 5′ non-translated regions of salivary gland and liver α-amylase mRNAs, has no counterpart in Amy-2^a. Some introns exhibit considerable sequence homology, suggesting that Amy-1^a and Amy-2^a have evolved by duplication from a common split ancestor sequence. Repetitive sequence elements occur in the introns and flanking regions of these genes. Gene titration by quantitative autoradiography reveals only one copy of Amy-1^a, but two copies of Amy-2^a per haploid mouse genome. In addition to Amy-1^a and Amy-2^a, several other amylase-like DNA sequences exist in the mouse genome. No gross rearrangements of amylase DNA sequences can be detected between germline DNA and that of various mouse tissues.     

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.