Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

Isolation and characterization of calmodulin genes from Xenopus laevis.

Two cDNAs derived from Xenopus laevis calmodulin mRNA have been cloned. Both cDNAs contain the complete protein-coding region and various lengths of untranslated segments. The two cDNAs encode an identical protein but differ from each other by 5% nucleotide substitutions. The 5' and 3' untranslated regions, to the extent available, are highly homologous between the two cDNAs. The predicted sequence of X. laevis calmodulin is identical to that of vertebrate calmodulins from mammals and chickens and shows one substitution compared with electric eel calmodulin. Genomic DNA sequences homologous to each of the two cDNA clones have been isolated and were shown to account for the major calmodulin-coding DNA sequences in X. laevis. These data suggest that X. laevis carries two active, nonallelic calmodulin genes. Although no complete analysis has been carried out, it appears that the X. laevis calmodulin genes are interrupted by at least four introns. The relative concentrations of calmodulin mRNA have been estimated in different embryonic stages and adult tissues and found to vary by up to a factor of 10. The highest levels of calmodulin mRNA were found in ovaries, testes, and brains. In these three tissues, the two calmodulin genes appear to be expressed at approximately equal levels.     

Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs.

Two genomic DNAs with a size of approximately 2.8 kb, isolated from the liver of Bungarus multicinctus (Taiwan banded krait), encode the precursors of the long neurotoxins, alpha-Bgt(A31) and alpha-Bgt(V31), respectively. Both genes share virtually identical overall organization with three exons separated by two introns, which were inserted in the same positions in the coding regions of the genes. Moreover, their nucleotide sequences share approximately 98% identity. This result indicates that the two genes co-exist in the genome of B.multicinctus, and probably arose from gene duplication. The exon/intron structures of the alpha-Bgt genes were essentially the same as those reported for the short neurotoxins. This reflects that the long and short neurotoxins should share a common evolutionary origin. Comparative analyses on long neurotoxin and short neurotoxin genes showed that the protein coding regions of the exons were more diverse than the introns except for the signal peptide domain. This implies that the protein coding regions of the neurotoxins may have evolved via accelerated evolution. PCR amplification of venom gland cDNA mixtures revealed that only two amino acid sequences corresponding to alpha-Bgt(A31) and alpha-Bgt(V31) could be deduced from the cDNAs. The results of chromatographic analyses and protein sequencing again emphasized the view that, with the exception of alpha-Bgt(A31) and alpha-Bgt(V31), no other alpha-Bgt isotoxins with amino acid substitutions were present in B.multicinctus venom. In contrast to the proposition of Liu et al. ( Nucleic Acids Res., 1998,26, 5624-5629), our findings strongly suggest that each alpha-Bgt isotoxin is derived from the respective gene, and that alpha-Bgt RNA polymorphism does not originate from one single, intronless gene by the mechanism of RNA editing.     

The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.     

Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

cDNA libraries of chicken spleen and Harder gland (a gland enriched with immunocytes) constructed in pBR322 were screened by differential hybridization and by mRNA hybrid-selected translation. Eleven L-chain cDNA clones were identified from which VL probes were prepared and each was annealed with kidney DNA restriction digests. All VL probes revealed the same set of bands, corresponding to about 15 germline VL genes of one subgroup. The nucleotide sequences of six VL clones showed greater than or equal to 85% homology, and the predicted amino acid sequences were identical or nearly identical to the major N-terminal sequence of L-chains in chicken serum. These findings, and the fact that the VL clones were randomly selected from normal lymphoid tissues, strongly indicate that the bulk of chicken L-chains is encoded by a few germline VL genes, probably much less than 15 since many of the VL genes are known to be pseudogenes. Therefore, it is likely that somatic mechanisms operating prior to specific triggering by antigen play a major role in the generation of antibody diversity in chicken. Analysis of the constant region locus (sequencing of CL gene and cDNAs) demonstrate a single CL isotype and suggest the presence of CL allotypes.     

<Emphasis Type="Italic">Oscheius tipulae</Emphasis> as an Example of eEF1A Gene Diversity in Nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.     

Characterization of a stress-induced,developmentally regulated gene family from soybean

We describe a family of stress-induced, developmentally regulated soybean genes for which cDNAs have been obtained from two different cultivars (Glycine max cv. Mandarin and Glycine max cv. Williams). The mRNAs corresponding to these cDNAs, called SAM22 and H4, respectively, accumulate predominantly in the roots of soybean seedlings but are present at high levels in the roots and leaves of mature plants. SAM22 accumulation is especially dramatic in senescent leaves. In addition, SAM22 accumulation can be induced in young leaves by wounding or by transpiration-mediated uptake of salicylic acid, methyl viologen, fungal elicitor, hydrogen peroxide or sodium phosphate (pH 6.9). Taken together, these data indicate that the genes corresponding to SAM22 and H4 are induced by various stresses and developmental cues. Southern blot analysis indicates that multiple copies of sequences related to SAM22 exist in the soybean genome. We also show that the nucleotide sequences of the cDNAs corresponding to SAM22 and H4 are 86% identical at the nucleotide level to each other and 70% identical at the amino acid level to the disease resistance response proteins of Pisum sativum.     

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.     

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.     

Intron-containing tRNA genes ofSulfolobus solfataricus

Summary Nucleotide sequences of four tRNA genes from the archaebacteriumSulfolobus solfataricus have been determined. Based upon DNA sequence analysis, three of the four genes contain presumptive intervening sequences (introns) in their anticodon loops. The three introns can form similar, but not identical, secondary structures. The cleavage site at the 3 end of all three introns occurs in a three-base bulge loop. All four genes lack an encoded 3 CCA terminus and are flanked by A+T-rich DNA sequences. Two of the genes are located on antiparallel DNA strands, with their 3 termini separated by 414 bp of sequence. Including two previously published sequences, a total of five introns have now been detected among sixS. solfataricus tRNA genes. Occurrence of introns at corresponding locations in both archaebacterial and eukaryotic tRNA genes suggests that the intron/exon form of gene structure predates the evolutionary divergence of the archaebacteria and the eukaryotes.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Molecular Characterization of Duplicate Cytosolic Phosphoglucose Isomerase Genes in Clarkia and Comparison to the Single Gene in Arabidopsis

The nucleotide sequence of PgiC1-a which encodes a cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.