Putative Full-length cDNA Research Articles

Identification and expression analysis of the gene lhcSR associated with adaptation to light and low temperature stress in the green tide forming alga Ulva prolifera

As the dominant species of the green tide-forming algae in the Yellow Sea of China during the past four years, Ulva prolifera was proposed as having evolved a perfect response mechanism to deal with different light and temperature stress. The LHCSR (also known as LI818 in green algae and LHCX in diatoms) protein is a stress-related member of the LHC family found only in algae. The homologues in the green alga Chlamydomonas were up-regulated in response to a variety of stresses and required for energy quenching. In this study, we cloned a putative lhcSR full-length cDNA sequence from U. prolifera and analysed the expression in response to various temperature and illumination gradients. The results showed that expression of lhcSR was up-regulated by light and transiently accumulated in 1 h. Expression of lhcSR could also be induced by low-temperature stress and could sustain the up-regulation for up to 6 days. These data suggest that the lhcSR gene in U. prolifera is involved in the adaption to the light and low-temperature stress in both the short and long term.

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the “resistant” population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity.

KLC1-ALK: A Novel Fusion in Lung Cancer Identified Using a Formalin-Fixed Paraffin-Embedded Tissue Only

The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5′-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

Structural and functional characterization of S-adenosylmethionine (SAM) synthetase from Pichia ciferrii

S-adenosylmethionine synthetase (SAM-s) catalyzes the synthesis of S-adenosylmethionine (SAM), which is essential for methylation, transcription, proliferation, and production of secondary metabolites. Here SAM-s from Pichia ciferrii were selectively cloned using RNA CapFishing and rapid amplification of cDNA ends (RACE). The putative full-length cDNA of SAM-s encoded a 383 amino acid protein (42.6 kDa), which has highly conserved metal binding sites, a phosphate-binding site, and functionally important motifs. The corresponding enzyme was over-expressed in a heterologous host of Pichia pastoris, and then purified to a homogenous form. Enzyme kinetics, immunoblotting, circular dichroism (CD), high performance liquid chromatography (HPLC), and molecular modeling were conducted to characterize the SAM-s from P. ciferrii. Structural and functional studies of SAM-s will provide important insights for industrial applications.

Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences

BackgroundWith the availability of rice and sorghum genome sequences and ongoing efforts to sequence genomes of other cereal and energy crops, the grass family (Poaceae) has become a model system for comparative genomics and for better understanding gene and genome evolution that underlies phenotypic and ecological divergence of plants. While the genomic resources have accumulated rapidly for almost all major lineages of grasses, bamboo remains the only large subfamily of Poaceae with little genomic information available in databases, which seriously hampers our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies.ResultsHere we report the cloning and sequencing of 10,608 putative full length cDNAs (FL-cDNAs) primarily from Moso bamboo, Phyllostachys heterocycla cv. pubescens, a large woody bamboo with the highest ecological and economic values of all bamboos. This represents the third largest FL-cDNA collection to date of all plant species, and provides the first insight into the gene and genome structures of bamboos. We developed a Moso bamboo genomic resource database that so far contained the sequences of 10,608 putative FL-cDNAs and nearly 38,000 expressed sequence tags (ESTs) generated in this study.ConclusionAnalysis of FL-cDNA sequences show that bamboo diverged from its close relatives such as rice, wheat, and barley through an adaptive radiation. A comparative analysis of the lignin biosynthesis pathway between bamboo and rice suggested that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content to reduce pollutants generated from bamboo pulping.

The cotton ATP synthase δ1 subunit is required to maintain a higher ATP/ADP ratio that facilitates rapid fibre cell elongation

The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthase activity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors of ATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation.

Comparative EST transcript profiling of peach fruits under different post-harvest conditions reveals candidate genes associated with peach fruit quality

BackgroundCold storage is used to inhibit peach fruit ripening during shipment to distant markets. However, this cold storage can negatively affect the quality of the fruit when it is ripened, resulting in disorders such as wooliness, browning or leathering. In order to understand the individual and combined biological effects that factors such as cold storage and ripening have on the fruit and fruit quality, we have taken a comparative EST transcript profiling approach to identify genes that are differentially expressed in response to these factors.ResultsWe sequenced 50,625 Expressed Sequence Tags (ESTs) from peach mesocarp (Prunus persica O'Henry variety) stored at four different postharvest conditions. A total of 10,830 Unigenes (4,169 contigs and 6,661 singletons) were formed by assembling these ESTs. Additionally, a collection of 614 full-length and 1,109 putative full-length cDNA clones within flanking loxP recombination sites was created.Statistically analyzing the EST population, we have identified genes that are differentially expressed during ripening, in response to cold storage or the combined effects of cold storage and ripening. Pair-wise comparisons revealed 197 contigs with at least one significant difference in transcript abundance between at least two conditions. Gene expression profile analyses revealed that the contigs may be classified into 13 different clusters of gene expression patterns. These clusters include groups of contigs that increase or decrease transcript abundance during ripening, in response to cold or ripening plus cold.ConclusionThese analyses have enabled us to statistically identify novel genes and gene clusters that are differentially expressed in response to post-harvest factors such as long-term cold storage, ripening or a combination of these two factors. These differentially expressed genes reveal the complex biological processes that are associated with these factors, as well as a large number of putative gene families that may participate differentially in these processes. In particular, these analyzes suggest that woolly fruits lack the increased boost of metabolic processes necessary for ripening. Additionally, these results suggest that the mitochondria and plastids play a major role in these processes. The EST sequences and full-length cDNA clones developed in this work, combined with the large population of differentially expressed genes may serve as useful tools and markers that will enable the scientific community to better define the molecular processes that affect fruit quality in response to post-harvest conditions and the organelles that participate in these processes.

Molecular Cloning and Characterization of a Novel Gossypium hirsutum L. bHLH Gene in Response to ABA and Drought Stresses

The phytohormone ABA was known to play a vital role in modulating plant responses to drought stress, and AtMYC2 (a GhbHLH1 homolog) had been proved to act as transcriptional activator involved in the dehydration and ABA response pathway in Arabidopsis. Here, using AtMYC2 amino acid sequence as a querying probe to screen Gossypium hirsutum L. EST database and contigging the candidate ESTs, the putative full-length cDNA sequence of cotton bHLH transcription factor was assembled and confirmed by amplifying leaves cDNA of drought-treated upland cotton cv Jinmian 19. The full-length cDNA named as GhbHLH1 has an open-reading frame of 2,025 bp, encoding a protein of 674 amino acids with a calculated molecular mass of 73.6 kDa and an isoelectric point of 5.46. Sequence alignment shows that GhbHLH1 contains a 60 amino acid long bHLH domain and have high homology with bHLH domain proteins in Arabidopsis, especially with AtMYC2, which plays an important role in response to stress stimuli. Semi-quantitative RT-PCR reveals that GhbHLH1 is strongly expressed in 7-day- post anthesis fibers but weak in roots, stems, and leaves. Based on real-time quantitative RT-PCR, the expression of GhbHLH1 in leaves was transitorily induced by ABA and PEG treatments, although its transcripts were accumulated in various organs. However, its expression was not affected by salt and cold treatments. Our results may provide the basis for future research of bHLH domain gene’s roles as a regulator of ABA signaling in cotton.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5' RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59-72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F(2) population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Transcriptome analysis of human gastric cancer

To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

A novel Arnt-interacting protein Ainp2 enhances the aryl hydrocarbon receptor signaling

In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCΔ418). Two clones, namely Ainp1 and Ainp2 ( Arnt- interacting peptide), were identified and subsequently Ainp2 was further characterized. Ainp2 interacts with TH-ArntCΔ418 in the GST pull-down and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells at the mRNA and protein levels. Results from the transient transfection studies using a DRE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 expression showed that Ainp2 enhances the 3-methylchloranthrene-induced activity in HepG2 cells, suggesting that Ainp2 plays a role in the Arnt-dependent function.

Isolation and characterization of a cDNA clone of UDP-glucose: anthocyanin 5- O-glucosyltransferase in Iris hollandica

UDP-glucose: anthocyanin 5- O-glucosyltransferase (5GT) catalyzes the transfer of the glucosyl moiety from UDP-glucose to the 5-hydroxyl group of anthocyanin. A putative full-length cDNA encoding 5GT was isolated from a cDNA library from flower buds of Iris hollandica by screening with a cDNA clone of UDP-glucose: anthocyanidin 3- O-glucosyltranseferase from Antirrhinum majus as a probe. The cDNA encodes an open reading frame of 463 amino acids with a calculated molecular mass of 50,100 Da. Heterologous expression of the cDNA in Escherichia coli demonstrated that it encoded 5GT. This is the first report of 5GT gene cloning from monocotyledonous plants. A molecular phylogenetic analysis of the amino acid sequences of glycosyltransferases from various plants showed that Ih5GT is a member of the 5GT group, although distant from dicot subgroup.

Isolation and characterization of chondrolectin ( Chodl), a novel C-type lectin predominantly expressed in muscle cells

In this study, we identified and characterized the mouse orthologue of the human chondrolectin gene, Chodl. Chodl is located at chromosome 16C3 and consists of six exons and five introns. The putative full-length mouse cDNA of Chodl consists of 2393 bp, with an open reading frame of 839 bp, 243 bp of 5′ untranslated region and 1310 bp of 3′ untranslated region. The predicted Chodl protein is a type I transmembrane protein containing one carbohydrate recognition domain (CRD) of C-type lectin in its extracellular portion and shares a significant similarity (45%) with layilin, a hyaluronan receptor. Reverse transcription–polymerase chain reaction and subsequent Southern blotting analysis revealed that in adult mice, Chodl is preferentially expressed in skeletal muscle, testis, brain, and lung. Analysis of the embryonic expression of Chodl showed that during gestation (embryonic day (E) 7–15) its expression is up-regulated. In situ hybridization on E15 mouse embryo revealed that Chodl is expressed in muscle cells of heterogeneous origin, including those from tongue, trunk, and tail. Furthermore, fluorescent immunostaining on limbs of newborn mice, localized the Chodl protein to striated muscle cells. Finally, Western blot analysis demonstrated expression of Chodl protein during the proliferation as well as differentiation phases of the myoblastic C2C12 cell line.

完全長ｃＤＮＡとゲノム医科学

As the result of the human genome project, the entire sequence of human genome was determined. Unfortunately, it is still not a trivial task to identify genes from these genome sequences only by the computational methods. Thus, full-length cDNAs are indispensable for the identification of genes and the determination of their structures. In addition, cDNAs will be a valuable resource for the functional analysis of the gene. We have performed so called FLJ project of collecting and determining the entire sequences of putative full-length cDNA clones. In this project, we extensively used cDNA libraries made by oligo-capping method. In this review, I will describe the current status of FLJ project and high-through-put functional analysis using full-length cDNA clones obtained by FLJ project.

A single-base deletion in soybean flavonoid 3'-hydroxylase gene is associated with gray pubescence color.

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3'-hydroxylase (F3'H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3'H gene. A putative full-length cDNA, sf3'h1 was isolated by 3' and 5' RACE. Sequence analysis revealed that sf3'h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3'H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3'H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3'h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3'h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the T locus in a F2 population segregating for the T locus. The above results strongly suggest that sJ3'h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.

A Mammalian Gene Evolved from the Integrase Domain of an LTR Retrotransposon

FIG. 1.—Summary of the structure and coding sequence of the human Gin-1 gene. Sequences of human cDNAs with accession numbers XMp003947.2 (a putative full-length cDNA), BE502574, AW173201.1, AW950418.1, AI631948.1, and AA766836.1 were used to deduce and confirm these data. The full-length protein is 522 amino acids long. The Gin-1 coding region spans nucleotides 36153–15345 in the genomic clone NTp002663.4. Arrowheads and the numbers above them, respectively, indicate the positions and lengths of introns. Several Alu repeats were detected within the two largest introns. Bold letters indicate the region homologous to the most conserved part of the IN domain, detailed in figure 2 and used to obtain the tree shown in figure 3. Amino acids characteristic of the H2C2 and DDE motifs (Khan et al. 1991) and of the most conserved region of the GPY/F module (Malik and Eickbush 1999) are underlined. Ty3/Gypsy long-terminal-repeat (LTR) retrotransposons are among the best-known transposable elements. They inhabit the genomes of many eukaryotic organisms, such as slime molds, plants, fungi, and animals, including vertebrates (Xiong and Eickbush 1990; Malik and Eickbush 1999; Miller et al. 1999; Marin and Llorens 2000). However, in spite of extensive genomic information, these elements had never been found in mammals. In the process of building a database of integrase domain (IN) sequences, we found an intriguing human sequence very similar to the IN of Ty3/Gypsy elements. It was particularly similar to the IN of the Drosophila melanogaster 412 element (E value 5 10227). The sequence of the human gene, which we called Gypsy integrase-1, or Gin-1, was reconstructed by combining information from genomic and cDNA sequences present in the National Center for Biotechnology Information databases (online at http://www.ncbi.nlm.nih.gov/; sequences in TIGR and Sanger Center databases did not provide additional information). Partial mouse, rat, and cow orthologous cDNAs were also detected. Moreover, an apparently full-length mouse cDNA sequence (accession number AK015243) was also found. However, the corresponding genomic sequences are not yet available for any of these other mammalian species. Figure 1 summarizes the structure and describes the protein encoded by the human Gin-1 gene. It has the characteristic H2C2, DDE, and GPY/F motifs found in many retroviral and retrotransposon integrases (Khan et al. 1991; Malik and Eickbush 1999). Homology to IN of Ty3/Gypsy elements spans the whole protein sequence (fig. 2), strongly suggesting that GIN-1 is also, and exclusively, an integrase. The similarity between the human and mouse genes is the expected similarity for orthologous genes of these two species. Amino acidic identity is 446/552 5 85%, while a comparative analysis of 1,138 mouse/human orthologs estimated an average identity in their coding regions of 86.4% (Makalowski and Boguski 1998). Phylogenetic analyses using IN sequences of the known clades of Ty3/Gypsy elements (Malik and Eickbush 1999), as well as of representative Ty1/Copia elements and retroviruses, confirmed that the putative integrase encoded by Gin-1 is very similar to Mdg1 clade elements IN (this clade so far includes D. melanogaster 412 and Mdg1; Malik and Eickbush 1999). Their sequences form a strongly supported monophyletic group

OCP1, an F-box protein, co-localizes with OCP2/SKP1 in the cochlear epithelial gap junction region

Immunohistochemical data indicate that OCP1 co-localizes exactly with OCP2 in the epithelial gap junction region of the guinea pig organ of Corti (OC). Despite the abundance of OCP1 in the OC, gaining access to its coding sequence – and, in particular, the 5′ end of the coding sequence – proved unexpectedly challenging. The putative full-length OCP1 cDNA – 1180 nucleotides in length – includes a 67 nucleotide 5′ leader sequence, 300 codons (including initiation and termination signals), and a 216 nucleotide 3′ untranslated region. The cDNA encodes a protein having a predicted molecular weight of 33 700. The inferred amino acid sequence harbors an F-box motif spanning residues 52–91, consistent with a role for OCP1 and OCP2 in the proteasome-mediated degradation of select OC proteins. Although OCP1 displays extensive homology to an F-box protein recently cloned from rat brain (NFB42), clustered sequence non-identities indicate that the two proteins are transcribed from distinct genes. The presumptive human OCP1 gene was identified in the human genome databank. Located on chromosome 1p35, the inferred translation product exhibits 94% identity with the guinea pig OCP1 coding sequence.

Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita.

Amplified fragment length polymorphism fingerprinting of three pairs of Meloidogyne incognita near-isogenic lines (NILs) was used to identify markers differential between nematode genotypes avirulent or virulent against the tomato Mi resistance gene. One of these sequences, present only in the avirulent lines, was used as a probe to screen a cDNA library from second-stage juveniles (J2s) and allowed cloning of a cDNA encoding a secretory protein. The putative full-length cDNA, named map-1, encoded a 458 amino acid (aa) protein containing a predictive N-terminal secretion signal peptide. The MAP-1 sequence did not show any significant similarity to proteins deposited in databases. The internal part of the protein, however, was characterized by highly conserved repetitive motives of 58 or 13 aa. Reverse transcription polymerase chain reaction (RT-PCR) experiments confirmed that map-1 expression was different between avirulent and virulent NILs. In PCR reactions, map-1-related sequences were amplified only in nematode populations belonging to the three species against which the Mi gene confers resistance: M. arenaria, M. incognita, and M. javanica. Polyclonal antibodies raised against a synthetic peptide deduced from the MAP-1 sequence strongly labeled J2 amphidial secretions in immunofluorescence microscopy assays, suggesting that MAP-1 may be involved in the early steps of recognition between (resistant) plants and (avirulent) nematodes.

The Genomic Organization of Type I Keratin Genes in Mice

We isolated two new keratin cDNAs by screening a cDNA library constructed from poly(A)+RNA of the dorsal and abdominal skin of C57BL/10J mice with a probe of humanKRT14.Due to its high sequence homology to human keratin 17 cDNA, one full-length cDNA is most likely to be mouse keratin 17 (Krt1-17) cDNA. The other is the putative full-length cDNA of a novel type I keratin gene, designatedKrt1-c29.These two keratin genes were mapped to the distal portion of Chromosome 11, where the mouse keratin gene complex-1 (Krt1) is localized. To elucidate the genomic organization ofKrt1in mice, we carried out genetic and physical analyses ofKrt1.A large-scale linkage analysis using intersubspecific backcrosses suggested that there are two major clusters inKrt1,one containingKrt1-c29, Krt1-10,andKrt1-12and the other containingKrt1-14, -15, -17,and-19.Truncation experiments with two yeast artificial chromosome clones containing the two clusters above have revealed that the gene order ofKrt1is centromere–Krt1-c29–Krt1-10–Krt1-12–Krt1-13–Krt1-15–Krt1-19–Krt1-14–Krt1-17–telomere. Finally, we analyzed sequence divergence between the genes belonging to theKrt1complex. The results clearly indicated that genes are classified into two major groups with respect to phylogenetic relationship. Each group consists of the respective gene cluster demonstrated by genetic and physical analyses in this study, suggesting that the physical organization of theKrt1complex reflects the evolutionary process of gene duplication of this complex.

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus.

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.