A new member of the CAB gene family: structure,expression and chromosomal location of Cab-8, the tomato gene encoding the Type III chlorophyll a/b-binding polypeptide of photosystem I

We have previously reported the isolation and characterization of tomato nuclear genes encoding two types of chlorophyll a/b-binding (CAB) polypeptides localized in photosystem (PS) I and two types of CAB polypeptides localized in PSII. Sequence comparisons shows that all these genes are related to each other and thus belong to a single gene family. Here we report the isolation and characterization of an additional member of the tomato CAB gene family, the single tomato nuclear gene, designated Cab-8, which encodes a third type of CAB polypeptide localized in PSI. The protein encoded by Cab-8 is 65% and 60% divergent from the PSI Type I and Type II CAB polypeptides, respectively. The latter two are 65% divergent from each other. Only some short regions of the polypeptides are strongly conserved. The Cab-8 locus maps to chromosome 10, 9 map units from Cab-7, the gene encoding the Type II PSI CAB polypeptide. The Cab-8 gene contains two introns; the first intron matches in position the single intron in the Type II PSII CAB genes and the second intron matches in position the second intron in the Type II PSI CAB gene. Like other CAB genes, Cab-8 is light-regulated and is highly expressed in the leaf and to a lesser extent in other green organs.     

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.     

The rapidly phosphorylated 25 kDa polypeptide of the light- harvesting complex of photosystem II is encoded by the type 2 cab-II genes

The main light-harvesting complex of Photosystem II (LHC II) in higher plants consists of two sub-populations. The 'inner' pool consists only of a 27 kDa polypeptide, whereas in the 'outer' pool both the 27 kDa and a 25 kDa polypeptide are found. We purified the 25 and the 27 kDa LHC II polypeptides from Scots pine and 25 kDa LHC II polypeptide from spinach. Protein sequencing after cleavage with endoproteinase Lys-C showed that the 25 kDa polypeptide is encoded by the Type 2 cab-II genes and the 27 kDa polypeptide by the Type I cab-II genes. A fatty acid was not covalently attached to the peptides assembled into the pigment-protein complex. Our results show that the different polypeptides seen on a gel are different gene products, and not the result of different processing.     

Nucleotide sequence and chromosomal location of Cab11 and Cab12, the genes for the fourth polypeptide of the photosystem I light-harvesting antenna (LHCI)

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned, cab11 and cab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome; cab11 on chromosome 3 and cab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chl a/b-binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma-exposed sequences preceding them. However, near the N-terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI-specific.     

Sequence of the fourth and fifth Photosystem II Type I chlorophyll a/b-binding protein genes of Arabidopsis thaliana and evidence for the presence of a full complement of the extended CAB gene family

A second locus (Lhb1B) encoding Photosystem II Type I chlorophyll a/b-binding (CAB) polypeptides was identified in Arabidopsis thaliana. This locus carries two genes in an inverted orientation. The predicted sequences of the polypeptides encoded by these two genes show substantial divergence in their amino termini relative to each other and to the proteins encoded by the three Lhb1 CAB genes previously characterized [10], but little divergence within the predicted primary structure of the mature protein. DNA probes derived from seven additional types of tomato CAB genes, encoding chlorophyll a/b-binding polypeptides of several antenna systems of the photosynthetic apparatus, were tested against A. thaliana. Each of these hybridized in Southern blots to unique DNA fragment(s), demonstrating the existence of each of these different types of CAB genes in the genome of A. thaliana. The number of genes encoding each CAB type in A. thaliana was estimated to be similar to that of tomato.     

Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley

We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.     

The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae.

DNA polymerase III (delta) of Saccharomyces cerevisiae is purified as a complex of at least two polypeptides with molecular masses of 125 and 55 kDa as judged by SDS-PAGE. In this paper we determine partial amino acid sequences of the 125 and 55 kDa polypeptides and find that they match parts of the amino acid sequences predicted from the nucleotide sequence of the CDC2 and HYS2 genes respectively. We also show by Western blotting that Hys2 protein co-purifies with DNA polymerase III activity as well as Cdc2 polypeptide. The complex form of DNA polymerase III activity could not be detected in thermosensitive hys2 mutant cell extracts, although another form of DNA polymerase III was found. This form of DNA polymerase III, which could also be detected in wild-type extracts, was not associated with Hys2 protein and was not stimulated by addition of proliferating cell nuclear antigen (PCNA), replication factor A (RF-A) or replication factor C (RF-C). The temperature-sensitive growth phenotype of hys2-1 and hys2-2 mutations could be suppressed by the CDC2 gene on a multicopy plasmid. These data suggest that the 55 kDa polypeptide encoded by the HYS2 gene is one of the subunits of DNA polymerase III complex in S.cerevisiae and is required for highly processive DNA synthesis catalyzed by DNA polymerase III in the presence of PCNA, RF-A and RF-C.     

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     

Identification of Lhcb gene family encoding the light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

The Lhcb gene family in green plants encodes several light-harvesting Chl a/b-binding (LHC) proteins that collect and transfer light energy to the reaction centers of PSII. We comprehensively characterized the Lhcb gene family in the unicellular green alga, Chlamydomonas reinhardtii, using the expressed sequence tag (EST) databases. A total of 699 among over 15,000 ESTs related to the Lhcb genes were assigned to eight, including four new, genes that we isolated and sequenced here. A sequence comparison revealed that six of the Lhcb genes from C. reinhardtii correspond to the major LHC (LHCII) proteins from higher plants, and that the other two genes (Lhcb4 and Lhcb5) correspond to the minor LHC proteins (CP29 and CP26). No ESTs corresponding to another minor LHC protein (CP24) were found. The six LHCII proteins in C. reinhardtii cannot be assigned to any of the three types proposed for higher plants (Lhcb1-Lhcb3), but were classified as follows: Type I is encoded by LhcII-1.1, LhcII-1.2 and LhcII-1.3, and Types II, III and IV are encoded by LhcII-2, LhcII-3 and LhcII-4, respectively. These findings suggest that the ancestral LHC protein diverged into LHCII, CP29 and CP26 before, and that LHCII diverged into multiple types after the phylogenetic separation of green algae and higher plants.     

Isolation of two different subpopulations of the light-harvesting chlorophyll a/b complex of photosystem II (LHCII)

Isolated LHCII from spinach has been solubilized and fractionated by non-denaturing isoelectric focusing to yield two subpopulations with different polypeptide but equal chlorophyll composition. One LHCII subpopulation contains only a 27 kDa polypeptide while the other contains the 27 and 25 kDa polypeptides in about equal amounts. The polypeptide patterns of the two subpopulations closely correspond to those suggested previously for the inner LHCII and peripheral LHCII, respectively.     

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina

Two clones have been isolated from a genomic library of the moss Physcomitrella patens and a cDNA library of the halotolerant green alga Dunaliella salina. The isolates contain genes coding for the major light-harvesting chlorophyll-a/b-binding protein (CAB) in the photosystem II (PSII) light-harvesting complex (LHCII). The 2544-bp insert of the moss genomic clone contains the complete CAB-coding region and 5' and 3' flanking sequences. The coding region contains an intron of 359 bp which is spanned by a pair of 9-bp perfect direct repeats. There are two CCAAT boxes and five enhancer-like elements related to (G)TGGTTTAAA(G) (Weiher et al., 1983) residing in the intron. Comparisons of the moss cab gene with sequences of light-inducible genes of higher plants reveal homologous and repeated sequences similar to the enhancer element in the 5' region upstream from the TATA and CCAAT boxes thought to be responsive to light inducibility. The 1256-bp algal cDNA contains the complete CAB-coding sequence, a 170-bp 5'-nontranslated region, and a 264-bp 3'-nontranslated region. While the overall homology in the nontranslated regions is low between the cab gene of the moss and that of the alga, the 3'-nontranslated regions of the two contain some sequences that are conserved among the cab genes in higher plants. The deduced amino acid sequences of these two clones are highly conserved except for the N-terminal region. Their hydropathic plots are very similar and both possess three hydrophobic segments that are likely alpha-helical transmembrane segments. The proposed CAB transit peptide sequence of the alga is divergent from that of the moss or higher plants, suggesting that they may have evolved from different origins. Southern blot analysis shows that the cab genes in the moss and the alga, as in higher plants, are encoded by a number of homologous genes constituting a multigene family.     

叶绿素缺乏大麦突变体光系统I色素蛋白复合物的研究

同野生型大麦相比,突变大麦光系统ＩＩ捕光色素蛋白复合物的含量明显降低,其多肽组分也发生了变化：２６ｋＤ的多肽缺失,２４ｋＤ、２７ｋＤ和３０ｋＤ多肽含量减少。ＲＮＡ印迹杂交结果表明突变大麦中ｃａｂ基因的表达与野生大麦基本一致,说明突变大麦中ＬＨＣＩ多肽的缺乏不是在转录水平引起的障碍,而很可能是受转录后水平的调节。突变大麦叶绿体的内膜系统处于发育的初级阶段,基粒较少,类囊体膜的垛叠也受到很大的限制,这可能与２６ｋＤ多肽的缺失有关。     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.