The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1

Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Ras superfamily (G1-G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sar1 family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S. cerevisiae and that they may perform similar biological functions.     

Nature and regulation of pistil-expressed genes in tomato

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.     

Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract

Two genes for Ca2+-dependent protein kinases, PCaPK-alpha and PCaPK-beta, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK-alpha encoded 481 amino acids and that of PCaPK-beta encoded 493 amino acids, predicting molecular masses of 55603 Da and 57131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK-alpha and PCaPK-beta were closely related to those of the Ca2+-dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin-dependent protein kinase family (35-48% identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C-terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30-34% identity). Two Ca2+-dependent protein kinases previously characterized from Paramecium (52 kDa CaPK-1, and 50 kDa CaPK-2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+-binding motifs encoded in PCaPK-alpha and PCaPK-beta, and the immunological cross-reaction of expressed cloned fragments of these genes with CaPK-2, suggest that they encode proteins of the same family.     

Schlafen, a new family of growth regulatory genes that affect thymocyte development

The Schlafen (Slfn) family of genes are differentially regulated during thymocyte maturation and are preferentially expressed in the lymphoid tissues. Ectopic expression of the prototype member Slfn1 early in the T lineage profoundly alters cell growth and development. In these mice, the DP thymocytes fail to complete maturation, and, depending on the transgene dosage, the number of thymocytes is reduced to 1%-30% of normal. Furthermore, expression of the Schlafen family members in fibroblasts and thymoma cells either retards or ablates cell growth. The conceptual protein sequences deduced for each of the family members have no similarity to characterized proteins and must therefore participate in a heretofore unknown regulatory mechanism guiding both cell growth and T cell development.     

The identification and localization of a human gene with sequence similarity to Polycomblike of Drosophila melanogaster

The Drosophila Polycomb group (PcG) of genes is required for the epigenetic regulation of a number of important developmental genes, including the homeotic (Hox) genes. The members of this gene family encode proteins that do not share sequence similarity, implying that each plays a unique role in this epigenetic repression mechanism. Polycomblike (Pcl) was the second PcG gene to be identified. We report here the isolation and characterization of a human cDNA, termed PHF1, which encodes a protein with significant sequence similarity to Drosophila Polycomblike (PCL). The region of similarity between PHF1 and PCL includes the two PHD fingers (C4-H-C3 motif), the region between them, and sequences C-terminal to the PHD fingers. PHF1 and PCL are 34% identical over this 258-residue region. PHF1 was mapped to 6p21.3 by fluorescence in situ hybridization. While several genetic diseases that are likely to result from developmental abnormalities map to this region, PHF1 is not a clear candidate gene for any of them.     

Differential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. oryzae

Induction of peroxidase has been correlated with resistant interactions between rice and Xanthomonas oryzae pv. oryzae. To assist in analysis of the role of rice peroxidases in plant defense against the bacterial pathogen, three peroxidase genes, POX22.3, POX8.1, and POX5.1, were identified from a rice cDNA library that was constructed from leaves of plants undergoing a resistant reaction. These genes were highly similar in nucleic acid and amino acid sequences and belonged to a gene family. The three genes showed differential expression in infiltrated rice leaves during pathogen interactions and mechanical stress. Only two peroxidase genes, POX8.1 and POX22.3, were predominantly expressed during resistant interactions. These two genes also were expressed during susceptible interactions, but induction was delayed compared with resistant interactions. POXgX9, a fourth peroxidase gene that was isolated from a genomic library, is adjacent to POX22.3 in the rice genome and has greater than 90% similarity in nucleotide and amino acid sequence identity to POX22.3. Interestingly, POXgX9 was expressed only in the roots of rice plants. While POX22.3 was expressed in both leaves and roots, POX8.1 and POX5.1 were not detected in roots but were induced in leaves by mechanical wounding at different times after treatment. POX22.3, POX8.1, and POX5.1 were estimated to be present in single copies in rice haploid genome. These results indicate that different members of the rice peroxidase gene family are distinctly regulated in response to various environmental cues.     

Analysis of xylem formation in pine by cDNA sequencing

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.     

Assembly of the zygotic centrosome in the fertilized Drosophila egg

Large-scale cDNA analysis provides several great advantages for genome investigations in rice. Isolated and partially characterized cDNA clones have contributed not only to the construction of an RFLP linkage map and physical maps of the chromosomes but also to investigations of the mechanisms of expression of various isozymes and family genes. The ultimate aim of our large-scale cDNA analysis is to catalogue all the expressed genes of this important cereal, including tissue-specific, developmental stage-specific, and stress-specific genes. As of August 1996, the Rice Genome Research Program (RGP) has isolated and partially sequenced more than 29,000 cDNA clones from various tissues and calluses in rice (Nipponbare, a japonica variety). The sequence data were translated into amino acid sequences for the 3 possible reading frames, and the similarity of these amino acid sequences to known proteins registered in PIR were examined. About 25% of the clones had significant similarities to known proteins. Some of the hit clones showed library-specific distributions, indicating that the composition of the clones in each library reflects, to some extent, the regulation of gene expression specific to differentiation, growth condition, or environmental stress. To further characterize the cDNA clones, including unknown clones, nucleotide sequence similarities of 24,728 clones were analyzed and the clones were classified into around 10,000 independent groups, suggesting that around a half or one third of expressed genes in rice have already been captured. These results obtained from our large-scale cDNA analysis provide useful information related to gene expression and regulation in rice.     

Experiences of mothers in five countries whose child died of cancer

The purpose of this study was to identify differentially expressed genes in normal and nitrofen-induced hypoplastic lungs in fetal mice. Such genes may play a role in the regulation of lung development. CD-1 pregnant dams were gavaged with 25 mg of nitrofen on gestational day (Gd) 8 to induce pulmonary hypoplasia and diaphragmatic hernia (DH). Normal and nitrofen-treated fetuses were removed on Gd 14 and Gd 16. Lungs were examined in all nitrofen-exposed fetuses and only those that had developed severely hypoplastic lungs with coexistent diaphragmatic hernia were taken for molecular analyses. RNA was extracted from normal and nitrofen-treated lungs, reverse transcribed, and PCR-amplified using 48 combinations of anchor and arbitrary primers for each condition. The resulting cDNAs from normal and hypoplastic lungs were run on 6% polyacrylamide differential display gels. In Gd 14 lungs, we observed 10 differentially expressed cDNA bands, of which 6 were identified to be inhibited and 4 were reduced in the hypoplastic lungs compared to normal fetal lungs. From the Gd 16 lungs, a total of 29 differentially expressed cDNA bands were found, of which 11 were reduced, 4 were inhibited, 11 were enhanced, and 3 were induced in the hypoplastic compared to the normal lungs. All 39 differentially expressed cDNAs were cloned, sequenced, and identified through BLAST searches. Among the sequences that were identified, results were as follows: 1) Hypoplastic Gd 14 lungs had two unknown cDNA sequences with reduced/inhibited expressions, whereas one was a known sequence having 77% similarity with a promoter region regulating various cytokines such as IL-1, IL-2, and IL-11. The expression of this sequence was inhibited in the hypoplastic lungs. This sequence also had similarity to lipid-binding proteins. 2) On Gd 16, hypoplastic lungs had one cDNA sequence with reduced expression which had 82% similarity with thyroid hormone receptor gene exon 1 and two other cDNA sequences with enhanced expressions. One of these enhanced cDNA sequences in hypoplastic lungs had 98% similarity with the fibroblast growth factor receptor-3 gene, and the other was an unknown sequence. Northern blot hybridizations were performed to confirm the differential expression of the two sequences of interest, which were identified as thyroid hormone receptor and fibroblast growth factor (FGF) receptor-3. Overall, out of a total of 39 RT-PCR products (i.e., cDNAs), the abundance of which was altered by nitrofen, 6 were found to be homologous to sequences in Gen Bank through BLAST searches. These 6 sequences became the products of interest, and 3 of these 6 products were similar to previously identified genes. Our results may shed some light on regulatory aspects of lung development and open avenues for treatment of hypoplastic lungs and other respiratory problems in human neonates.