Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

The evolution of proteins from random amino acid sequences: II. Evidence from the statistical distributions of the lengths of modern protein sequences

This paper continues an examination of the hypothesis that modern proteins evolved from random heteropeptide sequences. In support of the hypothesis, White and Jacobs (1993, J Mol Evol 36:79–95) have shown that any sequence chosen randomly from a large collection of nonhomologous proteins has a 90% or better chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. The goal of the present study was to investigate the possibility that the random-origin hypothesis could explain the lengths of modern protein sequences without invoking specific mechanisms such as gene duplication or exon splicing. The sets of sequences examined were taken from the 1989 PIR database and consisted of 1,792 super-family proteins selected to have little sequence identity, 623 E. coli sequences, and 398 human sequences. The length distributions of the proteins could be described with high significance by either of two closely related probability density functions: The gamma distribution with parameter 2 or the distribution for the sum of two exponential random independent variables. A simple theory for the distributions was developed which assumes that (1) protoprotein sequences had exponentially distributed random independent lengths, (2) the length dependence of protein stability determined which of these protoproteins could fold into compact primitive proteins and thereby attain the potential for biochemical activity, (3) the useful protein sequences were preserved by the primitive genome, and (4) the resulting distribution of sequence lengths is reflected by modern proteins. The theory successfully predicts the two observed distributions which can be distinguished by the functional form of the dependence of protein stability on length.The theory leads to three interesting conclusions. First, it predicts that a tetra-nucleotide was the signal for primitive translation termination. This prediction is entirely consistent with the observations of Brown et al. (1990a,b, Nucleic Acids Res 18:2079–2086 and 18: 6339-6345) which show that tetra-nucleotides (stop codon plus following nucleotide) are the actual signals for termination of translation in both prokaryotes and eukaryotes. Second, the strong dependence of statistical length distributions on sequence-termination signaling codes implies that the evolution of stop codons and translation-termination processes was as important as gene splicing in early evolution. Third, because the theory is based upon a simple no-exon stochastic model, it provides a plausible alternative to a limited universe of exons from which all proteins evolved by gene duplication and exon splicing (Dorit et al. 1990, Science 250:1377–1382).     

The evolution of protein sequences by repetitious gene duplication: Clostridial flavodoxin

Summary Internal regularities of amino acid sequences of flavodoxins, FMN-containing, low molecular weight flavoproteins, were statistically examined using the minimum mutation method. The sequence ofClostridium pasteurianum flavodoxin shows statistically significant evidence of repetitious internal gene duplications at different levels of structure. Peptide pairs with a low chance probability of occurrence were frequently observed at a shift of 5 residues. The pairs with the lowest chance probabilites are a pair of heptapeptides at positions 39–45 vs. 44–50, a 5 residue shift (p = 9 × 10^–6). Most of the related pairs are consistent and could best be explained by the repeating pentapeptide sequence: (Lys-Gly-Ala-Asp-Val-)_n and appropriate gaps. Internal repetitions with longer shifts were also suggested for other flavodoxins. Repetitious gene duplication is proposed for the early stages of flavodoxin evolution.     

Globin and linker sequences of the giant extracellular hemoglobin from the leech Macrobdella decora

A detailed electrospray ionization mass spectrometric study of the 3.5-MDa hexagonal bilayer hemoglobin (HBL Hb) from the pond leech Macrobdella decora has shown it to consist of at least six 17-kDa globin chains, of which two are monomeric and the remaining four occur as disulfide-bonded heterodimers, and three 24-kDa nonglobin linker chains (Weber et al., J. Mol. Biol. 251: 703–720, 1995). The cDNA sequences of the five major constituent chains, globin chains IIA, IIB, B, and C and linker chain L1, are reported here. The globins and linkers share 30%–50% and 20%–30% identity, respectively, with other annelid sequences. Furthermore, IIB and C align with strain A of annelid sequences, whereas IIA and B align with the strain B sequences. Although chains B and C are monomeric, chains IIA and IIB form the main disulfide-bonded dimer. They also have some unusual features: the distal His (E7) is replaced by Phe in IIA, and the highly conserved CD1Phe is replaced by Leu in IIB. In spite of these unusual features, the functional properties of Macrobdella Hb are comparable to those of other HBL Hbs. A phylogenetic analysis of the globin sequences from Macrobdella, the polychaete Tylorrhynchus, the oligochaete Lumbricus, and the vestimentiferan Lamellibrachia, indicates that the two strains originated by gene duplication followed by additional duplication of each of the two strains. The mutation rate of the linkers appeared to be faster than that of the globin chains. The phylogenetic trees constructed using the Maximum Likelihood, Neighbor-Joining and Fitch methods showed the Macrobdella globin sequences to be closest to Lumbricus, in agreement with a view of annelid evolution in which the divergence of the polychaetes occurred before the divergence of the leeches from oligochaetes.     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

Epigenetic silencing may aid evolution by gene duplication

Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151–1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from pseudogenization. Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication– degeneration–complementation) process. Dedicated to the memory of Susumu Ohno     

Chromosome number evolution in the tribeBrassiceae (Brassicaceae): Evidence from isozyme number

Variation in isozyme number was used to assess the evolution of haploid chromosome numbers (n=6–75) and systematic relationships in the tribeBrassiceae, which is believed to be one of the few monophyletic tribes in theBrassicaceae. Ten enzyme systems were surveyed among 108 species in 35 genera of tribeBrassiceae and for 11 species from seven other tribes. The data indicated that taxa with n=7–13 and n=14–18 were similar in isozyme number, suggesting that genera with n=14–18 did not arise from polyploidy (i.e. entire duplication) of the n=7–13 genomes. These results suggest that aneuploidy and/or chromosome fusion/splitting have played a more significant role than polyploidy in the evolution of higher base chromosome numbers in the tribe. The detection of widespread isozyme duplication in the tribe is consistent with reports of extensive gene duplication in theBrassica crop species, and suggests that the common ancestor of the tribe already had undergone a polyploid event, i.e. complete genome duplication, prior to aneuploid divergence. Inheritance studies conducted onSinapis arvensis showed that segregation ratios at seven loci (Fbp-2,Gpi-2,Idh-2,Pgm-2,Pgm-2,Tpi-1,Tpi-1) conformed to those expected under Mendelian inheritance. Isozyme duplications were phylogenetically informative at various taxonomic levels in the tribe. In particular, duplications for cytosolic phosphoglucomutase (Pgm-2,Pgm-2) and plastid triosephosphate isomerase (Tpi-1,Tpi-1) were evident in 33 of the 35 genera examined, supporting the monophyletic status of theBrassiceae with the inclusion ofOrychophragmus and the exclusion of controversial membersCalepina andConringia.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.     

Highly Variable Polymorphism of the α-Amylase Gene Family in <Emphasis Type="Italic">Litopenaeus</Emphasis><Emphasis Type="Italic">vannamei</Emphasis> (Crustacea Decapoda)

-Amylase from the tropical shrimp Litopenaeus vannamei presents a high degree of polymorphism and at least eight different electromorphs are detected by electrophoresis. Based on nucleotide sequences, three cDNAs have been previously characterized. In this paper we report on the organization and the evolution of corresponding -amylase genes, determined after PCR amplification. Three AMY genes have been characterized, spanning over 3.3 kb and encoding mature proteins of 495 amino acids (aa), which are all expressed in the digestive gland. The existence of nine short introns, ranging from 86 to 454 bp, located at the same positions for each of the different genes, and presenting no similarity between them, is reported. Between 11 and 15% of changes are observed in the coding aa sequences of genes II and III compared to the gene I sequence respectively. One 5 putative promoter sequence has been sequenced and shows no classical TATA box upstream to the coding sequence. Based on the intron size difference, a single PCR (producing the S–R fragments) allows the separation of a partial gene I (750 bp), corresponding to cDNA 20, from the others (650–680 bp). Sequencing different S–R PCR fragments from one shrimp shows at least eight different haplotypes. A complex microsatellite repeat is present in intron 6 of gene II. Using size and sequence differences in this repeated portion, it is possible to characterize two gene subfamilies (IIa and IIb) encoding previously described cDNAs 28 and 37, respectively. For the gene II family, two to four alleles are present in one shrimp corresponding to these two genes. Within the Panama natural population, 35 different alleles are shown at this locus. Regarding -amylase gene structure in the shrimp, many recombinants are present from a set of individuals and constitute an important mechanism of evolution of -amylase function. Accession numbers: AJ132379, L. vannamei -amylase gene I; AJ133526, gene II; AJ133119, gene III     

Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/GUS-gene fusion

Summary The 5-upstream region of the class I patatin gene B33 directs strong expression of the -glucuronidase (GUS) reporter gene in potato tubers and in leaves treated with sucrose. Cis-acting elements affecting specificity and level of expression were identified by deletion analysis in transgenic potato plants. A putative tuber-specific element is located downstream from position –195. Nuclear proteins present in leaf and tuber extracts bind specifically to a conserved AT rich motif within this region. A DNA fragment between –183 and –143, including the binding site is, however, not able to enhance the expression of a truncated 35S promoter from cauliflower mosaic virus. Independent positive elements contributing to a 100-fold increase relative to the basic tuber-specific element are located between –228 and –195; –736 and –509, –930 and –736 and –1512 and –951. Sucrose inducibility is controlled by sequences downstream of position –228, indicating that the tuber-specific and sucrose-inducible elements are in close proximity.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

Interdomain linkage in the polymeric hemoglobin molecule of Artemia

Artemia has evolved the longest known concatenation of hemoglobin domains, the subunit containing nine domains and the subunit having a similar size. Translation of the cDNA sequence of the subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit.The interdomain linker sequences were 14%–64% identical (62%–91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.The position of interdomain introns and the character of a variable residue early in the linker are compatible with the nine-domain polymer having evolved through gene duplication reflected in globin domain fusion incorporating an extension specifically of the N-terminus. The multiplication of an original single-domain globin gene to give the present nine is estimated from sequence differences, allowing for multiple mutations at individual sites, to have occurred in a period at least 500–700 million years ago.Correspondence to: C.N.A. Trotman 1444     

Examination of protein sequence homologies: V. New perspectives on evolution between bacterial and chloroplast-type ferredoxins inferred from sequence evidence

Summary Sequence homologies among 34 chloroplast-type ferredoxins were examined using a computer program that quantitatively evaluates the extent of sequence similarity as a correlation coefficient. The resultant alignment contains six gaps representing insertions or deletions of some residues, all of which are located such that they precisely preserve the domains of structural fragments as determined by crystallographic data onSpirulina platensis ferredoxin.In the search for any total correlation between the chloroplast-type and 27 bacterial ferredoxins, 1891 comparison matrices prepared for possible combinations indicated that the bacterial basal sequence of 55 residues has been conserved evolutionarily in the chloroplast-type sequences corresponding to residue positions 36–90 ofSpirulina platensis ferredoxin. In addition, the bacterial connector sequence region was found to be conserved. These findings strongly suggest that the bacterial and chloroplast-type ferredoxins descended from a common ancestor, and branched off after the bacterial gene duplication, whereas the chloroplast-type ferredoxins originally were generated by duplicating the already duplicated bacterial gene, i.e., by double-duplication.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.     

The evolution of muscular parvalbumins investigated by the maximum parsimony method

Summary Phylogenetic trees requiring the lowest sum of nucleotide replacements and gene duplicative events were constructed from the amino acid sequence data on ten gnathostome parvalbumins (PAR) and two related myofibrillar proteins troponin-C (TNC) and myosin alkali-light-chain (ALC). The origin and differentiation of the structural domains within these proteins were also investigated by the maximum parsimony method and by an alignment statistic for identifying evolutionarily related protein sequences. The results suggest, in agreement with the Weeds-McLachlan model, that tandem duplications in a precursor gene caused a primordial one-domain polypeptide (consisting of two helices with a calcium binding region in between) to double and then quadruple in size. Duplications of the gene coding for this four domain (I–II–III–IV) protein in an early metazoan, pre-gnathostome lineage gave rise to the separate loci for TNC, ALC, and PAR. TNC, which alone retained the Ca-binding function in each of its four domains, evolved much more slowly than either the ALC or PAR lineages. In the PAR lineage the I–II–III–IV structure was degraded, presumably by a partial gene deletion, to the II–III–IV structure during descent to the gnathostome ancestor of parvalbumins. Also during this period the mid region in domain II lost its Ca-binding function and, as it did so, evolved at an accelerated rate over other regions, a pattern indicative of positive selection for a change in function. In turn, from the gnathostome ancestor to the present, the mid regions of domains III and IV, which each retained Ca-bindung function, evolved much more slowly than other regions, a pattern indicative of stabilizing selection for preservation of function. Between the gnathostome and teleost-tetrapod ancestor a gene duplication separated the parvalbumins into an-lineage and a-lineage. During this early vertebrate period PAR genes evolved at the extremely fast rate of 89 nucleotide replacements per 100 codons per 10⁸ years (i.e. 89 NR %), but from the teleost-tetrapod ancestor to the present, both- and-PAR lineages evolved at a much slower rate, about 8 NR %. The use of-parvalbumins as phylogenetic markers was complicated by presumptive evidence that paralogous (i.e. duplication dependent) gene lineages occur within this group. As a final point, in the genealogy of TNC, ALC, and PAR lineages, a non-random pattern of nucleotide replacements was observed between the reconstructed ancestral and descendant mRNA sequences. The pattern was similar to that observed for other protein genealogies and seems to reflect a bias in the genetic code for guanine to adenine and adenine to guanine transitions (especially at the first nucleotide position of the RNA codons) to produce amino acid substitutions which are compatible with the preservation of protein three-dimensional structure.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Covalent Structure of Botulinum Neurotoxin Type E: Location of Sulfhydryl Groups, and Disulfide Bridges and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without ^I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.