Expression of three ABA-regulated clones and their relationship to maturation processes during the embryogenesis of chick-pea seeds

Water stress inhibits germination of chick-pea seeds and produces specific changes in gene expression. some of which are coincident with those induced by the exogenous application of abscisic acid (ABA). Three cDNA clones, GAB-8, GAB-9 and GAB-11, were previously identified as under the regulation of ABA and osmotic stress in embryonic axes of germinating chick-pea. Here we try to establish a relationship between the changes in gene expression induced by ABA and stress conditions during germination and those naturally occurring during the desiccation process that leads to seed maturation. Our results show that the germinative capacity of chick-pea is related to the water content of the organ. In vitro translation of the mRNAs from developing seed reveals that in the later stages of seed maturation some polypeptides appear that previously were found to be regulated by ABA and by water deficit in germinating seeds. Hybridization by northern blot of embryogenic mRNAs with GAB-8. GAB-9 and GAB-11 clones shows that the mRNAs corresponding to such clones only appear in the later phases of seed formation, coinciding with seed dehydration, and persisting until seeds became fully mature. The results suggest that these mRNAs are probably related to the response to dehydration that occurs during seed maturation, and that the pattern of expression of these ABA-regulated clones coincides with that of the established late embryogenesis-abundant (LEA) genes.     

Rapid evolution of variants in a rodent multigene family encoding salivary proteins

A survey of polypeptides encoded by RNA isolated from the submandibular glands of members of the Muridae (species of Mus and Rattus), in conjunction with cDNA cloning, has identified a class of salivary proteins that we term "spot proteins." Although clearly homologous, these proteins show dramatic differences between species in their polypeptide length. On the basis of the sequence of the corresponding clones, it is inferred that the rat spot 1 protein has a size of 6,370 daltons (Da), whereas that of the inbred mouse spot 1 is 11,603 Da. A second component is expressed in some stocks and strains of Mus, and this spot 2 protein has a size of up to 19,212 Da. The sizes of the corresponding mRNAs show parallel differences, and the variation in the sizes of mRNAs in different species of Mus correlates with the pattern of speciation, the size increasing with increased relatedness to inbred mice. The spot protein sequence comprises three domains: an N-terminal domain rich in hydroxy and acidic amino acids, a central domain consisting of repeats of a 9-amino-acid sequence, and a C-terminal domain that in the mouse is very basic. Variation in the number of repeats largely accounts for the differences in size between the mouse and rat mRNAs and their encoded polypeptides, and the coding sequence appears to have been expanding during speciation in the Muridae. There is extensive divergence in sequence between the mouse and rat mRNAs and their encoded proteins. The pattern of amino acid replacements and nucleotide substitutions is consistent with little, if any, selection constraint on the precise sequence of the spot proteins, suggesting that it is the overall architecture of the molecule, rather than the precise structure, that is important for function. There is strong evidence for a gene conversion event having occurred between the two mouse sequences. Frequent recombination by unequal crossing-over between spot protein coding sequences, if it occurs between active and silent genes, could account not only for the expansion in their size but also for their rapid divergence.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.     

Gene expression patterns associated with in vitro tracheary element formation in isolated single mesophyll cells of Zinnia elegans.

Tracheary element formation from isolated Zinnia leaf mesophyll cells is an excellent system for the dissection of patterned secondary cell wall thickening and lignification. We used mRNAs from cells cultured for 48 h in the induction medium to isolate differentially regulated genes. Thirteen unique cDNA clones were isolated using a subtractive hybridization method. These clones can be divided into three distinct groups according to their characteristic gene expression in different media. The first group includes those genes whose expression is induced in the basal medium without 1-naphthaleneacetic acid (NAA) and benzyladenine; this indicates that the expression of these genes is regulated by chemical and physical factors other than these hormones. Three of these clones, p48h-229, p48h-114, and p48h-102, show significant homology to a pathogenesis-related protein II, a serine proteinase inhibitor, and a sunflower anther-specific proline-rich protein, respectively. The second group includes those genes whose expression is mainly NAA induced. One of these clones, p48h-10, shows high protein sequence homology to a barley aleurone-specific cDNA, B11E. The p48h-10-encoded protein shares some common characteristics of plant nonspecific lipid transfer proteins (low molecular weight, the secretion signal peptide, eight conserved cysteine residues, and a basic protein), although no significant protein sequence homology is found between p48-10 and other plant nonspecific lipid transfer proteins. The third group includes those genes whose expression is induced primarily in the induction medium; this indicates that the expression of these genes is closely associated with the process of tracheary element formation. Two of these clones, p48h-107 and p48h-17, show high homology to adenylate kinase and papaya proteinase I, respectively. The possible roles of these differentiation-specific genes during tracheary element formation are discussed.     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.     

Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS

Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The α-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their coding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins will enable the genetic mapping of cloned genes as well as the analysis of their translational regulation.     

Cloning of a cDNA encoding an Artemia franciscana Na/K ATPase alpha-subunit.

Clones of cDNA that code for an isoform of the Artemia franciscana Na/K ATPase alpha subunit (NaKA alpha) have been isolated. The sequence of the longest of these clones (pArATNa136) is 3595 nucleotides; it codes for a 1004-amino acid protein whose sequence is identical to that of two previously sequenced Artemia NaKA alpha peptides. The encoded protein is over 73% identical to Drosophila melanogaster and vertebrate NaKA alpha s, and 73.8% identical to another Artemia NaKA alpha isoform previously described (named alpha 2850 in this article). The two Artemia cDNA clones code for mRNAs of different size; the clone pArATNa136 codes for a 4.5-kb mRNA while the alpha 2850 clone codes for a 3.6-kb mRNA. The degree of homology and the different size of the mRNAs encoded by both cDNAs suggest that they code for two different isoforms of the protein.     

A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic α-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.     

Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison

A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA.     

Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes

Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N). In the present study we investigated whether human B and T lymphocytes recognize these conserved regions. The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area. Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide. A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N. The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M. Four clones that recognized the more amino-terminal fragment also responded to infected E. According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.     

Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family

We analyzed five malate synthase cDNA clones from the higher plant Brassica napus L. We determined the complete mRNA sequence and showed that the longest cDNA clone, pMS1, contains the entire protein coding region. The deduced polypeptide consists of 561 amino acids with a molecular mass of 63,700 daltons. To discern whether the cloned mRNAs represent distinct malate synthase polypeptides, we compared restriction maps and partial nucleotide sequence of the cDNA clones as well as their pattern of hybridization with restriction fragments in nuclear DNA. The results suggest that the five cloned mRNAs are encoded by either a single gene or by highly conserved members of the gene family.