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.     

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.     

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

Identification of type 1 and type 2 light-harvesting chlorophyll a/b-binding proteins using monospecific antibodies.

The amino acid sequences of more than 40 apoproteins of the light-harvesting complex associated with Photosystem II (LHC II) of various plants have been deduced by sequencing their corresponding genes. These highly conserved sequences fall into two major categories, type 1 and type 2, that differ mainly in a small number of domains close to the N-terminus. We have made polyclonal, monospecific antibodies against synthetic peptides corresponding to the most unique sequence domains of the N-terminal regions of type 1 and type 2 LHC II apoproteins, using sequences derived from petunia genes. On Western blots our anti-type 1 and 2 antibodies crossreact with light-harvesting proteins of petunia, tomato, spinach and several other plants. By using a new gel-system based on ammediol (2-amino-2-methyl-1,3-propanediol), we are able to resolve up to eight LHC II apoproteins. On petunia, tomato and spinach blots the anti type 1 antibodies bind to two or more of the higher molecular weight LHC II polypeptides, whereas the anti type 2 antibodies recognize very specifically only one or two of the lower molecular weight LHC-proteins. In all plants studied, the type 1 LHC II apoproteins are more numerous and span a greater size range than the type 2 apoproteins. This is consistent with the smaller number of type 2 LHC II CAB genes that have been discovered to date.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Sequence homologies in the protamine gene family of rainbow trout

We have sequenced five different rainbow trout protamine genes plus their flanking regions. The genes are not clustered and do not contain intervening sequences. There is an extremely high degree of sequence conservation in the coding and 3' untranslated regions of the gene. Downstream sequences exhibit little homology though conserved regions are found 250 base pairs 3' to the gene. There are four regions upstream of the gene that are highly conserved in the six clones, including the canonical Goldberg - Hogness box which is 45 base pairs 5' to the coding region. A second homologous region is found 90 bases upstream. Although in the same approximate location as the CAAT box found upstream of other genes, it does not contain the canonical CAAT sequence. Further upstream of the protamine genes at -115 there is an A-T rich sequence while a 25 base pair conserved sequence is located 150 bases upstream. In addition we report the presence of a potential Z-DNA region of predominantly A-C repeats approximately one kilobase downstream of one of the genes.     

Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce

Resistance Gene Candidate2 (RGC2) genes belong to a large, highly duplicated family of nucleotide binding site-leucine rich repeat (NBS-LRR) encoding disease resistance genes located at a single locus in lettuce (Lactuca sativa). To investigate the genetic events occurring during the evolution of this locus, approximately 1.5- to 2-kb 3' fragments of 126 RGC2 genes from seven genotypes were sequenced from three species of Lactuca, and 107 additional RGC2 sequences were obtained from 40 wild accessions of Lactuca spp. The copy number of RGC2 genes varied from 12 to 32 per genome in the seven genotypes studied extensively. LRR number varied from 40 to 47; most of this variation had resulted from 13 events duplicating two to five LRRs because of unequal crossing-over within or between RGC2 genes at one of two recombination hot spots. Two types of RGC2 genes (Type I and Type II) were initially distinguished based on the pattern of sequence identities between their 3' regions. The existence of two types of RGC2 genes was further supported by intron similarities, the frequency of sequence exchange, and their prevalence in natural populations. Type I genes are extensive chimeras caused by frequent sequence exchanges. Frequent sequence exchanges between Type I genes homogenized intron sequences, but not coding sequences, and obscured allelic/orthologous relationships. Sequencing of Type I genes from additional wild accessions confirmed the high frequency of sequence exchange and the presence of numerous chimeric RGC2 genes in nature. Unlike Type I genes, Type II genes exhibited infrequent sequence exchange between paralogous sequences. Type II genes from different genotype/species within the genus Lactuca showed obvious allelic/orthologous relationships. Trans-specific polymorphism was observed for different groups of orthologs, suggesting balancing selection. Unequal crossover, insertion/deletion, and point mutation events were distributed unequally through the gene. Different evolutionary forces have impacted different parts of the LRR.     

Correlation of apoproteins with the genes of the major chlorophyll a/b binding protein of photosystem II in Arabidopsis thaliana. Confirmation for the presence of a third member of the LHC IIb gene family

The major light-harvesting complex in higher plants is LHC IIb. The LHC IIb of Arabidopsis thaliana contains 2 pigment-binding apoproteins of 28 and 25 kDa. To determine the relationship between them and the LHC IIb gene family members, each protein was purified to homogeneity, subjected to direct protein sequencing, and the sequences compared with those deduced from LHC IIb genes in this organism. The 28 kDa protein is the product of Type I LHC IIb genes. The 25 kDa LHC IIb component is distinctly different from the 28 kDa LHC IIb protein, and is more closely related to the type III LHC IIb gene product of barley. Type III gene products lack the first 9-11 residues found in proteins of the Type I and II genes, a region that contains a phosphorylatable threonine residue. The lack of the N-terminal residues explains why this LHC IIb apoprotein has never been seen to be phosphorylated, and partly or wholly why it is smaller. The implication of the missing N-terminus on uptake of LHC II precursor proteins into the plastid and of the relative organization of the LHC IIb subunits in the PS II antenna is discussed.     

Immediate-early mRNA-2 of herpes simplex viruses types 1 and 2 is unspliced: conserved sequences around the 5' and 3' termini correspond to transcription regulatory signals

Nuclease S1 and exonuclease VII analyses of immediate-early (IE) mRNA-2 of herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) show them to be unspliced and of similar length. The DNA sequences around the 5' and 3' termini have been determined. Comparison of the sequences around the 5' ends reveals several common features. (1) Four discrete blocks of upstream homology which are precisely colinear with respect to the 5' termini of the mRNAs; the blocks include the 'TATA' box, a G-C rich sequence and a sequence (AATTAAATACAT) which may be involved in the coordinate induction of the IE class of genes. (2) Several copies of the sequence CCCCGCCC, found in different upstream positions in HSV-1 and HSV-2, which may be important in the expression of a wide variety of eukaryotic genes. (3) Potential hairpin structures in the region of the 5' termini which are present at similar locations in HSV-1 and HSV-2. Sequence comparison around the 3' termini of IEmRNA-2 reveals high homology at the proposed C-terminus of the polypeptide.