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Structural characterization of an alpha-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous alpha-amylase inhibitors.

The primary structures of two subunits of an alpha-amylase inhibitor (alpha AI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previously deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of alpha AI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that alpha AI-2 has the subunit stoichiometry of an alpha 2 beta 2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) alpha-amylase inhibitor, which is quite different in the inhibitory specificity from alpha AI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.

Amino Acid Sequence↗

Comparison of Sources and Lines Selected for Drought Resistance in Common Bean.

Drought is a major constraint to common bean (Phaseolus vulgaris L.) production worldwide. Our objectives were to (i) identify sources of drought resistant germplasm in common bean cultivars and (ii) compare drought resistant germplasm with lines selected from interracial and intergene pool populations. We included in this study 12 of the most promising drought resistant cultivars from race Durango and 11 from race Jalisco, nine drought resistant lines selected from interracial or intergene pool populations, and two drought resistant and two susceptible checks. The 36 genotypes were evaluated in drought-stressed (DS) and nonstressed (NS) environments in four cropping seasons between 1996 and 1998 at the International Center for Tropical Agriculture (CIAT), Palmira, Colombia. Drought stress reduced seed yield by 53%, 100-seed weight by 13%, and days to maturity by 3%. Race Durango cultivars had higher yield, larger seed weight, and earlier maturity than race Jalisco cultivars in DS and NS environments. Large variations within the two races were found for the three traits. Drought resistant selected lines out-yielded drought resistant checks by 44% in DS and 15% in NS and cultivars from race Durango by 48% in DS and 30% in NS and race Jalisco by 96% in DS and 46% in NS environments. Seed yield in DS was correlated negatively with the percent reduction (PR) because of drought stress and drought susceptibility index (DSI), whereas a positive correlation existed between PR and DSI. Drought resistant selected lines and race Durango cultivars had similar maturity. Mean 100-seed weight of selected lines (23 g) was less than race Durango (34 g) and race Jalisco cultivars (29 g). While new sources of drought resistance could be identified in races Durango and Jalisco, these drought resistant germplasm and selected lines derived from interracial and intergene pool populations should be utilized for improvement of drought resistance in common bean.

Journal Article↗

Serotype-specific detection of bean common mosaic potyvirus in bean leaf and seed tissue by enzymatic amplification.

An assay involving reverse transcription and polymerase chain reaction (PCR) is described for specific detection of serotypes A and B BCMV isolates in bean leaf and seed tissues. Three oligonucleotide primers designed according to the sequence data available allow, in appropriate combination, serotype-specific detection of BCMV. The sensitivity of the method was sufficient to detect BCMV in as little as 100 fg and 50 pg of infected leaf and seed tissues, respectively.

Base Sequence↗

A root-specific bZIP transcription factor is responsive to water deficit stress in tepary bean (Phaseolus acutifolius) and common bean (P. vulgaris).

Root cDNA libraries were differentially screened to isolate water deficit-responsive transcripts in the relatively drought-resistant plant tepary bean (Phaseolus acutifolius). A novel root-specific, water deficit-responsive transcript was identified and predicted to encode a bZIP transcription factor. The orthologous form of this gene was isolated from the drought-sensitive P. vulgaris and the patterns of expression of these genes compared. These genes have predicted amino acid sequences in the bZIP domain that are 64% similar to a soybean bZIP protein. There were three amino acid differences between the P. acutifolius bZIP and the P. vulgaris gene product. Both species transcribed this gene in a root-specific and water deficit-responsive manner. The cell-specific pattern of expression for the gene was determined using in situ hybridization and immunolocalization. Two tissues in the root accumulated the protein: epidermis and phloem. The nuclear localization of this protein was determined by electron microscopy. The bZIP protein accumulated in the nuclei of both the epidermal cell and the vascular cell in response to water deficit stress in both species in a similar manner.

Adaptation, Physiological↗

Serological and biological relationships among viruses in the bean common mosaic virus subgroup.

Bean common mosaic virus (BCMV), blackeye cowpea mosaic virus (B1CMV), cowpea aphid-borne mosaic virus (CABMV), azuki bean mosaic virus (AzMV), and peanut stripe virus (PStV) are five species of the genus Potyvirus, family Potyviridae which are seed-transmitted in beans or cowpeas. Eighteen isolates of BCMV, five isolates of B1CMV, four isolates of CABMV, and one isolate each of AzMV, and PStV were compared serologically using a panel of 13 monoclonal antibodies (MAbs) raised against BCMV, B1CMV, CABMV, or PStV in indirect enzyme-linked immunosorbent assay (ELISA). Four MAbs detected all virus isolates; one detected all isolates except those of CABMV. Three MAbs were specific only for serotype A isolates of BCMV. Four MAbs detected all serotype B isolates of BCMV plus all isolates of B1CMV, AzMV, and PStV. None of the antibodies distinguished among these four viruses. However, in biological tests with 11 bean cultivars selected for differentiating BCMV pathotypes, all isolates of B1CMV, AzMV, and PStV could be differentiated from the BCMV serotype B isolates by their reactions on a few bean cultivars in host group I and the cowpea cultivar California Blackeye #5. Potential problems that can arise from the use of nonauthenticated isolates are also discussed.

Antibodies, Monoclonal↗

Genetic mapping of the bean golden yellow mosaic geminivirus resistance gene bgm-1 and linkage with potyvirus resistance in common bean (Phaseolus vulgaris L.).

Bean golden yellow mosaic virus (BGYMV) is a whitefly-transmitted geminivirus of the Begomovirus family that causes important yield losses to common beans grown in tropical and sub-tropical countries of Latin America and the Caribbean. A major resistance gene that has been widely deployed in this region is the recessive locus bgm-1 that prevents the development of severe yellowing typical of the disease. In this study, we developed a co-dominant sequence-characterized amplified region (SCAR) marker, SR2, based on a previously identified random amplified polymorphic DNA (RAPD) marker that is tightly linked to the bgm-1 resistance gene and identified the position of the locus in the common bean genome through comparative mapping using two genetic maps for the species. The SR2 marker was mapped relative to bgm-1 in a segregating population of recombinant inbred lines developed from the resistant x susceptible cross of DOR476 x SEL1309. Polymorphism was shown to be based on a 37 bp insertion event in the SR2 allele associated with susceptibility compared to the allele associated with resistance and the marker mapped at a distance of 7.8 cM from the resistance gene. The SR2 marker was significantly associated with overall disease symptoms and with three of the four symptoms associated with the disease (yellowing or chlorosis, flower abortion, foliar deformation) in a greenhouse trial in Colombia with the mechanically transmissible BGYMV-Guatemala strain. In both the DOR364 x G19833 and BAT93 x Jalo EEP558 mapping populations, SR2 was located near the end of linkage group b03 (chromosome 5) suggesting a sub-telomeric position. The position of the bgm-1 resistance gene was estimated to be close to that of bc-1, a strain-specific resistance gene for Bean common mosaic virus (BCMV), based on linkage of SR2 with the SCAR marker SBD5 in the DOR364 x G19833 mapping population. The implications of linkage between these two recessive resistance genes are discussed, as this is the first association between resistance genes against both a begomovirus and a potyvirus.

Base Sequence↗

Effect of soaking prior to cooking on the levels of phytate and tannin of the common bean (Phaseolus vulgaris, L.) and the protein value.

The effect of soaking in domestic processing, on the nutritive value of the common bean (Phaseolus vulgaris, L.) cv IAC-Carioca, was studied. Five treatments were carried out with experimental diets, and offered to male, recently weaned Wistar rats. The protein sources were, respectively, control diet (casein) (CC), casein plus the soluble solids found in the soaking water (CSS), freeze dried bean cooked without soaking (BNS), freeze dried bean cooked with the non-absorbed soaking water (BSW), freeze dried bean cooked without the non-absorbed soaking water (BSNW). and an aproteic diet (AP) for corrective purposes. The anti-nutritional factors (phytates and tannins), were determined in the differently processed beans and in the soaking water. The following values for the reduction of phytates were obtained: BNS (20.9%), BSNW (60.8%) and BSW (53.0%), and the tannins were reduced by: BNS (86.6%), BSNW (88.7%) and BSW (89.0%). No significant differences were observed between the various treatments using the common bean as protein source, with respect to the net protein ratio (NPR). With respect to the digestibility corrected by non-protein diet, values varying between 94.1% and 94.6% for casein, and between 57.5% and 61.4% for the common bean, were observed, the treatment BNS being more digestible. It was concluded that soaking did not interfere with the NPR of the experimental diets containing the common bean as protein source, nor did it reduce the tannin content. However soaking was capable of reducing the phytate levels in the common bean. On the other hand, soaking was unable to increase the protein digestibility of the common bean, since the treatment BNS showed the highest value for digestibility.

Amino Acids↗

Sequencing and analysis of common bean ESTs. Building a foundation for functional genomics.

Although common bean (Phaseolus vulgaris) is the most important grain legume in the developing world for human consumption, few genomic resources exist for this species. The objectives of this research were to develop expressed sequence tag (EST) resources for common bean and assess nodule gene expression through high-density macroarrays. We sequenced a total of 21,026 ESTs derived from 5 different cDNA libraries, including nitrogen-fixing root nodules, phosphorus-deficient roots, developing pods, and leaves of the Mesoamerican genotype, Negro Jamapa 81. The fifth source of ESTs was a leaf cDNA library derived from the Andean genotype, G19833. Of the total high-quality sequences, 5,703 ESTs were classified as singletons, while 10,078 were assembled into 2,226 contigs producing a nonredundant set of 7,969 different transcripts. Sequences were grouped according to 4 main categories, metabolism (34%), cell cycle and plant development (11%), interaction with the environment (19%), and unknown function (36%), and further subdivided into 15 subcategories. Comparisons to other legume EST projects suggest that an entirely different repertoire of genes is expressed in common bean nodules. Phaseolus-specific contigs, gene families, and single nucleotide polymorphisms were also identified from the EST collection. Functional aspects of individual bean organs were reflected by the 20 contigs from each library composed of the most redundant ESTs. The abundance of transcripts corresponding to selected contigs was evaluated by RNA blots to determine whether gene expression determined by laboratory methods correlated with in silico expression. Evaluation of root nodule gene expression by macroarrays and RNA blots showed that genes related to nitrogen and carbon metabolism are integrated for ureide production. Resources developed in this project provide genetic and genomic tools for an international consortium devoted to bean improvement.

Carbon↗

Genetics of resistance to the geminivirus, Bean dwarf mosaic virus, and the role of the hypersensitive response in common bean.

Bean dwarf mosaic virus (BDMV) is a single-stranded DNA virus (genus: Begomovirus, family: Geminiviridae) that infects common bean ( Phaseolus vulgaris L.) and causes stunted plant growth, and mosaic and mottle symptoms in leaves. BDMV shows differential pathogenicity in common bean, infecting germplasm of the Andean gene pool (e.g., the snap bean cultivar Topcrop), but not that of the Middle American gene pool (e.g., the pinto bean cultivar Othello). Resistance to BDMV in Othello is associated with development of a hypersensitive response (HR) in vascular (phloem) tissues. In this study, Middle American germplasm representing the four recognized races (i.e., Durango, Guatemala, Jalisco, and Mesoamerica) and the parents of Othello were inoculated with BDMV and a BDMV-green fluorescent protein (GFP) reporter. All genotypes showed partial or complete resistance to BDMV and BDMV-GFP, indicating the widespread distribution of resistance in the Middle American gene pool. A number of BDMV-resistant germplasm did not show the HR, indicating it is not correlated with resistance. In the F(1), F(2), and F(3) of reciprocal crosses between Othello and Topcrop, a single dominant allele, Bdm, conferred BDMV resistance.

DNA, Viral↗

Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance.

Amino Acid Sequence↗

Crg, a gene required for Ur-3-mediated rust resistance in common bean, maps to a resistance gene analog cluster.

Race-specific resistance to the bean rust pathogen (Uromyces appendiculatus) is provided by a number of loci in common bean (Phaseolus vulgaris). The Ur-3 locus controls hypersensitive resistance (HR) to 44 of the 89 races curated in the United States. To better understand resistance mediated by this locus, we developed new genetic material for analysis. We developed a population of mutagenized seed of cv. Sierra (genotype = Ur-3 ur-4 ur-6) that was screened with a bean rust race that is normally incompatible (HR response) on Ur-3 genotypes. We discovered two mutants of common bean, crg and ur3-delta3, in which uredinia formed on leaves (a compatible interaction) following infection. The F1 generation from a cross of these two mutants expressed the HR response, and the F2 generation segregated in a ratio of 9:7 (HR/uredinia formation). Therefore, the two genes are unlinked. Further genetic analysis determined that the mutation in ur3-delta3 was in the Ur-3 locus, and the mutation in crg was in a newly discovered gene given the symbol Crg (Complements resistance gene). Each mutation was inherited in a recessive manner. Unlike ur3-delta3, crg expressed reduced compatibility to bean rust races 49 and 47 that are normally fully compatible on genotypes, such as Sierra, that are homozygous recessive at the Ur-4 and Ur-6 loci. This suggests a gene mutated in crg is normally a positive compatibility factor for the bean-bean rust interaction. Polymerase chain reaction analysis of crg with primers to common bean resistance gene analogs (RGA) that contain a nucleotide-binding site sequence similar to those found in a number of plant disease resistance genes revealed that crg is missing the SB1 RGA, but not the linked SB3 and SB5 RGAs. Genetic analyses revealed that Crg cosegregates with the SB1 RGA. These results demonstrate that Crg is located near a RGA cluster in the common bean genome.

Basidiomycota↗

A viral resistance gene from common bean functions across plant families and is up-regulated in a non-virus-specific manner.

Genes involved in a viral resistance response in common bean (Phaseolus vulgaris cv. Othello) were identified by inoculating a geminivirus reporter (Bean dwarf mosaic virus expressing the green fluorescent protein), extracting RNA from tissue undergoing the defense response, and amplifying sequences with degenerate R gene primers. One such gene (a TIR-NBS-LRR gene, RT4-4) was selected for functional analysis in which transgenic Nicotiana benthamiana were generated and screened for resistance to a range of viruses. This analysis revealed that RT4-4 did not confer resistance to the reporter geminivirus; however, it did activate a resistance-related response (systemic necrosis) to seven strains of Cucumber mosaic virus (CMV) from pepper or tomato, but not to a CMV strain from common bean. Of these eight CMV strains, only the strain from common bean systemically infected common bean cv. Othello. Additional evidence that RT4-4 is a CMV R gene came from the detection of resistance response markers in CMV-challenged leaves of RT4-4 transgenic plants, and the identification of the CMV 2a gene product as the elicitor of the necrosis response. These findings indicate that RT4-4 functions across two plant families and is up-regulated in a non-virus-specific manner. This experimental approach holds promise for providing insights into the mechanisms by which plants activate resistance responses against pathogens.

Cloning, Molecular↗

[Exudation and accumulation of citric acid in common bean in response to Al toxicity stress].

Significant differences in the exudation and accumulation of citric acid in common bean genotypes were observed in response to Al toxicity stress by hydroponic cultural experiments. Secreted citric acid increased with increasing external concentrations of Al3+ which ranged from 0 to 50 mumol.L-1, while ranged from 50 to 80 mumol.L-1, secreted citric acid decreased with increasing external concentrations of Al3+. Among different genotypic common beans, citric acid secreted in G19842 was the largest, while Al uptake per unit dry weight in G19842 was the least. No difference in the accumulation of citric acid in leaves was found among different genotypic common beans, while the size of the content of citric acid in roots was G19842 > AFR > ZPV > G5273. The amount of citric acid exuded was smaller induced by phosphorus deficiency than that induced by Al toxicity stress. Exposure to 50 mumol.L-1 LaCl3 could not induce the exudation of citric acid, and it implied that the exudation and accumulation of citric acid in common bean was an important physiological response of resistance to Al toxicity stress.

Aluminum↗

Hard-to-cook phenomenon in common beans--a review.

Legumes are one of the world's most important sources of food supply, especially in developing countries, in terms of food energy as well as nutrients. Common beans are a good source of proteins, vitamins (thiamine, riboflavin, niacin, vitamin B6) and certain minerals (Ca, Fe, Cu, Zn, P, K, and Mg). They are an excellent source of complex carbohydrates and polyunsaturated free fatty acids (linoleic, linolenic). However, common beans have several undesirable attributes, such as long cooking times, being enzyme inhibitors, phytates, flatus factors, and phenolic compounds, having a "beany" flavor, and being lectins and allergens, which should be removed or eliminated for effective utilization. Grain quality of common beans is determined by factors such as acceptability by the consumer, soaking characteristics, cooking quality, and nutritive value. Acceptability characteristics include a wide variety of attributes, such as grain size, shape, color, appearance, stability under storage conditions, cooking properties, quality of the product obtained, and flavor. Storage of common beans under adverse conditions of high temperature and high humidity renders them susceptible to a hardening phenomenon, also known as the hard-to-cook (HTC) defect. Beans with this defect are characterized by extended cooking times for cotyledon softening, are less acceptable to the consumer, and are of lower nutritive value. Mechanisms involved in the HTC defect have not been elucidated satisfactorily. Attempts to provide a definitive explanation of this phenomenon have not been successful. The most important hypotheses that have been proposed to explain the cause of bean hardening are (1) lipid oxidation and/or polymerization, (2) formation of insoluble pectates, (3) lignification of middle lamella, and (4) multiple mechanisms. Most researchers have reported that the defect develops in the cotyledons. Recently, some authors have suggested that the seed coat plays a significant role in the process of common bean hardening. A better knowledge of cotyledon and seed coat microstructure may lead to a better understanding of the causes of seed hardness. In order to prevent the development of the HTC defect several procedures have been proposed: (1) appropriate storage, (2) controlled atmospheres, and (3) pretreatments. Probably, the most workable solution to the hardening phenomenon may be the development of materials less prone to HTC phenomenon. Decreasing cooking time, increasing nutritive value, and improving sensory properties of seeds with HTC defect would have great nutritional and economical impact.(ABSTRACT TRUNCATED AT 400 WORDS)

Cooking↗

Development of a genome-wide anchored microsatellite map for common bean (Phaseolus vulgaris L.).

A total of 150 microsatellite markers developed for common bean ( Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican x Andean population, DOR364 x G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.

Base Sequence↗

Comparative bioinformatic analysis of genes expressed in common bean (Phaseolus vulgaris L.) seedlings.

To rapidly and cost-effectively generate gene expression data, we developed an annotated unigene database of common bean (Phaseolus vulgaris L.). In this study, 3 cDNA libraries were constructed from the bean breeding line SEL1308, 1 from young leaf and 2 from seedlings inoculated or not inoculated with the fungal pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, which causes anthracnose in common bean. To this date, 5255 single-pass sequences have been included in the database after selection based on sequence quality. These ESTs were trimmed and clustered using the computer programs Phred and CAP3 to form a unigene collection of 3126 unique sequences. Within clusters, 318 single nucleotide polymorphisms (SNPs) and 68 insertions-deletions (indels) were found, indicating the presence of paralogous gene families in our database. Each unigene sequence was analyzed for possible function using their similarity to known genes represented in the GenBank database and classified into 14 categories. Only 314 unigenes showed significant similarities to Phaseolus genomic sequences and P. vulgaris ESTs, which indicates that 90% (2818 unigenes) of our database represent newly discovered common bean genes. In addition, 12% (387 unigenes) were shown to be specific to common bean. This study represents a first step towards the discovery of novel genes in beans and a valuable source of molecular markers for expressed gene tagging and mapping.

Computational Biology↗

Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.

Amino Acid Sequence↗

Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-alpha-amylase inhibitor family of the common bean and its relatives.

The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and alpha-amylase inhibitor (alpha AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two alpha AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an alpha AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an alpha AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and alpha AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the alpha AIs. Proteolytic processing at an Asn-Ser site is required for the activation of alpha AI, and this site is present in all alpha AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur.

Amino Acid Sequence↗