Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.     

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30 kDa; serotype II, 38 kDa; serotype III, 34 kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores—< 51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

Evolutionary conservation of the chlorophyll a/b-binding proteins: cDNAs encoding type I, II and III LHC I polypeptides from the gymnosperm Scots pine

Summary cDNAs encoding three different LHC I polypeptides (Type I, Type II and Type III) from the gymnosperm Scots pine (Pinus sylvestris L.) were isolated and sequenced. Comparisons of the deduced amino acid sequences with the corresponding tomato sequences showed that all three proteins were highly conserved although less so than the LHC II proteins. The similarities between mature Scots pine and tomato Types I, II and III LHC I proteins were 80%, 87% and 85%, respectively. Two of the five His residues that are found in AXXXH sequences, which have been identified as putative chlorophyll ligands in the Type I and Type II proteins, were not conserved. The same two regions of high homology between the different LHC proteins, which have been identified in tomato, were also found in the Scots pine proteins. Within the conserved regions, the Type I and Type II proteins had the highest similarity; however, the Type II and Type III proteins also showed a similarity in the central region. The results suggest that all flowering plants (gymnosperms and angiosperms) probably have the same set of LHC polypeptides. A new nomenclature for the genes encoding LHC polypeptides (formerly cab genes) is proposed. The names lha and lhb are suggested for genes encoding LHC I and LHC II proteins, respectively, analogous to the nomenclature for the genes encoding other photosynthetic proteins.     

Characterization of the yellow-pigment genes of Erwinia herbicola

A 6.7 kb DNA fragment containing the pigment genes of Erwinia herbicola Eho13 has been cloned into Escherichia coli. These genes were chromosomally encoded in E. herbicola. The entire DNA fragment could be divided into at least three regions. Deletions in Region I resulted in a non-pigmented phenotype, a deletion in Region II resulted in a pink/yellow phenotype, deletions in Region III resulted in either a pink or a non-pigmented phenotype. Tn1000 insertions in the same regions, however, gave different phenotypes. Insertions in Region II produced a pink phenotype. Insertions in Region III resulted in either a light-yellow or a non-pigmented phenotype. Minicell studies showed that the 6.7 kb DNA fragment encoded at least five proteins (50 kDa, 42 kDa, 36 kDa, 35 kDa and 34 kDa). A 2.7 kb HindIII deletion in Region I caused the disappearance of these proteins, suggesting that this 2.7 kb fragment may play a regulatory role in pigment synthesis. Our results also showed that a 4.1 kb EcoRV fragment consisted of Region I and a part of Region II complemented a pink/yellow clone of Eho10 (pHL545), suggesting that the pigments of Eho13 and Eho10 were probably similar or identical.     

Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5''-upstream sequences.

Cytokeratins are a family of approximately 20 polypeptides which form the intermediate-sized filaments (IFs) characteristic of epithelial cells. They are synthesized co-ordinately as 'pairs' consisting of one representative from each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the more basic (type II) polypeptides, in cell type-specific combinations. We have isolated and characterized the genes coding for four bovine cytokeratins of the basic (type II) subfamily, i.e. cytokeratins Ib, III, IV and 6*, by Southern blot hybridization, hybridization-selection-translation experiments, hetero-duplex mapping, and partial sequencing of the exons coding for the hypervariable carboxy-terminal 'tail' regions of the proteins and the 3'-non-translated ends of the mRNAs which are distinct for the individual cytokeratin polypeptides. Limited 'chromosomal walk' experiments demonstrated that the genes are organized into two tandems, i.e. 6*----Ib and III----IV, in which they are separated by approximately 11 kb. RNA analysis by Northern and dot blots show that both genes of the III----IV tandem are co-expressed in some bovine tissues (muzzle epidermis, hoof pad and tongue mucosa) and cultured cells (BMGE + H) but that in other tissues, cornea for example, only the gene encoding III is expressed. Unexpectedly, the genes linked in the tandem 6*----Ib are not co-expressed in any of the tissues examined. mRNA from gene 6* has been found in tongue mucosa but in none of the other cell lines and tissues examined, whereas mRNA for cytokeratin Ib is expressed in cornea and muzzle epidermis but not in, for example, tongue mucosa and in the epidermis of the heel pad.(ABSTRACT TRUNCATED AT 250 WORDS)     

The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor-2 locus in barley (Hordeum vulgare L.)

Two-dimensional mapping (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the polypeptide components of “B” hordein fractions from eight barley varieties of widely different ancestry has been carried out. The relative positions of 47 different polypeptides were mapped, there being between 8 and 16 present in any one variety. The individual polypeptides differed in their distribution patterns; some were present in a number of varieties, while others were restricted to one or two. They also differed in their relative contributions to the total hordein fraction, both within and between varieties. The structural homology of the major polypeptides was compared by cleavage at methionine residues with cyanogen bromide and separation of the peptides on gradient gels. The polypeptides were classified into three groups which gave cleavage patterns with either two (class I), four (class II), or five (class III) low molecular weight bands. Class III polypeptides were found in all eight varieties, but in seven of the varieties class I or class II polypeptides were also present. With one exception, polypeptides migrating in the same position in different varieties gave identical or almost identical patterns. The three classes of polypeptides showed different distributions on the two-dimensional gels. Classes II and III polypeptides had a similar range of isoelectric points (pH 6.5–8.0), but all of the class II polypeptides were of slightly lower molecular weight. Class I polypeptides had a wider range of pI and molecular weight; the most alkaline and the lowest molecular weight polypeptides were in this group. The hordein fractions from a number of other barley varieties were compared with that of Julia. All had major polypeptides which migrated with ones present in Julia, but they differed in the relative amounts of these and in the absence of some polypeptides and the presence of others. B hordein is coded for by a single locus which has been suggested to be a complex multigenic family derived by duplication and divergence of a single gene. The data reported here provide support for this hypothesis and suggest that both mutations in the duplicated genes and recombination within the locus may have contributed to the polymorphism of the polypeptides.     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Molecular characterization of two clusters of genes encoding the Type I CAB polypeptides of PSII in Nicotiana plumbaginifolia

A Nicotiana plumbaginifolia genomic library in the phage Charon 34 was used to isolate and characterize 7 full-length genes and part of an 8th gene encoding chlorophyll a/b-binding (CAB) polypeptides. These genes are arranged in two clusters. All the genes within the clusters are arranged in opposite orientation to their neighbours. The nucleotide sequences of two genes, one from each cluster, show that both genes, designated Cab-E and Cab-C, encode very similar proteins (95.9% of homology) corresponding to type I photosystem II polypeptides. Southern blot analysis suggests that at least 19 CAB genes encoding type I PSII CAB polypeptides are present in the N. plumbaginifolia genome. We also describe the presence within the N. plumbaginifolia genome of CAB genes encoding PSII type II CAB polypeptides and PSI type I CAB polypeptides. The sequences of the 5 flanking region of three different CAB genes (Cab-E, Cab-C, and CAB-F) were determined. Two of them (Cab-C and Cab-F) share extensive homology, whereas the Cab-E promoter shows homology to Cab-C and Cab-F only in a unique region extending from the CAAT box to the TATA box. This conserved sequence is also found in the same position in promoters of CAB genes encoding type I PSII polypeptides from other plant species.Abbreviations CAB chlorophyll a/b-binding protein - bp base pair(s) - kb 1 000 bp     

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic α-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.     

The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion

ABSTRACT: BACKGROUND: Plant polyphenol oxidases (PPOs) are enzymes that typically use molecular oxygen to oxidize ortho-diphenols to ortho-quinones. These commonly cause browning reactions following tissue damage, and may be important in plant defense. Some PPOs function as hydroxylases or in cross-linking reactions, but in most plants their physiological roles are not known. To better understand the importance of PPOs in the plant kingdom, we surveyed PPO gene families in 25 sequenced genomes from chlorophytes, bryophytes, lycophytes, and flowering plants. The PPO genes were then analyzed in silico for gene structure, phylogenetic relationships, and targeting signals. RESULTS: Many previously uncharacterized PPO genes were uncovered. The moss, Physcomitrella patens, contained 13 PPO genes and Selaginella moellendorffii (spike moss) and Glycine max (soybean) each had 11 genes. Populus trichocarpa (poplar) contained a highly diversified gene family with 11 PPO genes, but several flowering plants had only a single PPO gene. By contrast, no PPO-like sequences were identified in several chlorophyte (green algae) genomes or Arabidopsis (A. lyrata and A. thaliana). We found that many PPOs contained one or two introns often near the 3' terminus. Furthermore, N-terminal amino acid sequence analysis using ChloroP and TargetP 1.1 predicted that several putative PPOs are synthesized via the secretory pathway, a unique finding as most PPOs are predicted to be chloroplast proteins. Phylogenetic reconstruction of these sequences revealed that large PPO gene repertoires in some species are mostly a consequence of independent bursts of gene duplication, while the lineage leading to Arabidopsis must have lost all PPO genes. CONCLUSION: Our survey identified PPOs in gene families of varying sizes in all land plants except in the genus Arabidopsis. While we found variation in intron numbers and positions, overall PPO gene structure is congruent with the phylogenetic relationships based on primary sequence data. The dynamic nature of this gene family differentiates PPO from other oxidative enzymes, and is consistent with a protein important for a diversity of functions relating to environmental adaptation.     

Genetic Diversity in Ralstonia solanacearum Strains from Mauritius using Restriction Fragment Length Polymorphisms

Race 1, biovar III of Ralstonia (synonym Pseudomonas ) solanacearum , causal organism of bacterial wilt, has been reported in Mauritius on several crops and plant species. The genetic relationship among 38 strains isolated from potato, tomato, bean and anthurium was determined by restriction fragment length polymorphisms (RFLPs). After hybridization with probe 5a67, five RFLP patterns could be distinguished. Types V and I were most commonly encountered. A common band of approximately 6.5 kb was found in 35 strains. Type I pattern consisted of only this band and was observed in 12 out of 16 anthurium strains tested. Type V was associated with 12 out of 16 potato strains and consisted of a band of approximately 3.3 kb in addition to the one observed in type I. RFLP patterns II, III and IV were less frequently encountered. The RFLP analysis showed that genetic diversity was present in race 1, biovar III strains. The relationship between the host and RFLP pattern is discussed.     

Differential utilization of beta-tubulin isotypes in differentiating neurites

beta-Tubulin is encoded in vertebrate genomes by a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different beta-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three beta-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with approximately 70% of types I and II assembled but only approximately 50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.     

Ribosomal DNA variation in foxtail millet, Setaria italica (L.) P. Beauv., and a survey of variation from Europe and Asia

 Restriction fragment length polymorphism (RFLP) and the structure of ribosomal RNA genes (rDNA) were investigated in 117 landraces of foxtail millet, Setaria italica (L.) P. Beauv. Five RFLP phenotypes were found when the genomic DNA was digested with BamHI; these were named types I–V. Of these types I, II and III were the most frequent. Type I was mainly distributed in the temperature zone, type II in the Taiwan-Philippines Islands and type III in South Asia. Restriction mapping of the cloned rDNA and comparison with RFLP phenotypes showed that the different types originated from a polymorphism in the length within the intergenic spacer (IGS) and BamHI site changes within the IGS. Received: 28 August 1996 / Accepted: 28 February 1997