Joakim Galli

Genetic analysis of non-insulin dependent diabetes mellitus in the GK rat

Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.

Design of an Optimized Scaffold for Affibody Molecules

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.

The Balbiani ring 3 gene in Chironomus tentans has a diverged repetitive structure split by many introns.

A set of approximately 15 secretory proteins is synthesized by the salivary gland cells in the midge Chironomus tentans. These proteins are secreted but do not form insoluble fibers until they are transported out of the gland lumen. A Balbiani ring (BR) gene family consisting of four genes (BR1, BR2.1, BR2.2 and BR6) have previously been shown to encode four of these proteins, sp-I a to d, with relative molecular weights of 1 x 10(6). Each BR gene contains an uninterrupted block in which about 100 repeats are tandemly arranged. The repeats are virtually identical and efficient homogenization mechanisms must operate within each block. Here we describe a new BR gene, the BR3 gene, which according to structural similarities may belong to the BR gene family, but at the same time exhibits a strikingly different structure. The gene encodes a 10.9 kb transcript that contains 38 introns and is spliced into a 5.5 kb mRNA. The mRNA is translated into a cysteine-rich 185 kDa major component of the gland secretion. The coding sequence in the gene is built from diverged repeats in which mainly the cysteine codons are preserved and the sequence is split by the introns into 17 to 678-bp long exons. The introns are located at defined positions in relation to the repeat structure. In sharp contrast to the uninterrupted array of identical repeats in the BR1-BR6 genes, the repeats in the BR3 gene are not efficiently homogenized and have diverged extensively from each other. We propose that the splitting of the repeat structure into variable sized exons prevents homogenizations dependent on unequal aligning of homologous sequences.

A new member of a secretory protein gene family in the dipteran Chironomus tentans has a variant repeat structure.

SummaryWe describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.

A rat genetic linkage map including 67 new microsatellite markers.

Genetic heterogeneity in outbred populations and a major impact of environmental factors on the disease phenotypes complicate genetic studies of multifactorial disorders. For control of these effects, inbred animal disease models are used for the genetic dissection of disease pathways that operate in concert to cause susceptibility to several human common disorders. Quantitative trait loci (QTLs) that contribute to disease phenotypes can be identified by analysis of experimental crosses between a strain with the disease phenotype of interest and a control strain in carefully controlled environments (Lander and Schork 1994). Several pathophysiologically well characterized inbred rat strains are developed as models for multifactorial disorders such as diabetes mellitus, inflammatory diseases, hypertension, and obesity (Greenhouse et al. 1990). In recent years, genome mapping of the rat has progressed with an increasing number of mapped genes and genetic markers (Bihoreau et al. 1997; Brown et al. 1998; Jacob et al. 1995; Pravenec et al. 1996), which has allowed genetic localization of QTLs in several rat models (Aitman et al. 1997; Galli et al. 1996; Gauguier et al. 1996; Hilbert et al. 1991; Jacob et al. 1991; Kanemoto et al. 1998; Kovacs and Kloting 1998). QTL identification and positional cloning of disease susceptibility genes rely on dense and accurate genetic maps, especially when new approaches with advanced intercross animals are used for highresolution genetic mapping (Darvasi 1998). In the present report, we describe the construction of a rat genetic linkage map based on anchor markers mapped in several hundred F2 progeny. The rats were derived from an intercross between the GK rat, a model for type 2 diabetes (Goto et al. 1975), and the F344 rat. Approximately 1200 microsatellite markers were PCR amplified, and 543 of these were informative in the GK/F344 cross and could be amplified readily. Most microsatellites were identified by screening genomic libraries and the Genebank database for repeated sequences (Jacob et al. 1995). Additional markers were also obtained from the Rat Map Database (http:// www.ratmap.gu.se/). GK and F344 rats were bred as described previously (Galli et al. 1996). Two reciprocal crosses (GK male × F344 female and F344 male × GK female) produced two types of F 1 progeny. The two sets of F1 progeny were intercrossed separately, and a total of 374 F2 progeny were obtained in two separate cohorts. We selected 45 F2 progeny (Galli et al. 1996) for genotyping with 400 informative microsatellites. Subsequently, 190 randomly selected rats from the two cohorts were genotyped with 194 of the 400 markers, spaced at an average of 10 cM. Finally, the remaining 139 rats were genotyped with 92 of the 194 markers to further improve resolution in previously identified QTLs (Galli et al. 1996). Rats were genotyped as previously described (Jacob et al. 1991), with the exception that one primer in each pair was labeled with g P-ATP. The linkage map was assembled by employing MAPMAKER/ EXP (version 3.0) linkage analysis software (Lander et al. 1987). The 400 markers were sorted into 27 separate linkage groups with a LOD of 5.0 as inclusion criterion. All linkage groups were asigned to specific chromosomes and oriented according to the rat cytogenetic map (Szpirer et al. 1998). All chromosomes except Chromosomes (Chrs) 6, 13, 15, and 16 were represented by single linkage groups. The linking process was subsequently repeated with a LOD of 4.0. This allowed mapping of all markers into 22 linkage groups. Each chromosome was represented by a single linkage group with the exception of Chr 13. The two linkage groups on Chr 13 were, however, linked with a LOD of 3.0. The resulting map consists of 400 markers in 21 linkage groups corresponding to the 20 autosomal chromosomes and the X Chr. This map includes 242 markers ordered with LOD

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans

3.0 and defined as framework (anchor) loci. The remaining 158 markers were placed according to their most likely positions in relation to the anchor markers. Considerable effort was made to assure accuracy in the mapping process: all genotypes were read twice, and all ambiguities were checked and resolved. The generated KI rat genetic linkage map spans a total length of 1782 cM (Kosambi 1943) ith an average spacing of consecutive markers of 4.4 cM (Table 1). Furthermore, the map includes 67 new markers, that is, 16 gene-specific markers and 51 anonymous microsatellites (Table 2). The map is available at http://www.ki.se/cmm. The sex-averaged genetic length of the map (1782 cM) is in

A new member of the Balbiani ring multigene family in the dipteran Chironomus tentans consists of a single-copy version of a unit repeated in other gene family members

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the spl2 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-by-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp 12 gene may represent a diverged member of the BR multigene family.

Identification of rat susceptibility loci for adjuvant-oil-induced arthritis

The known Balbiani ring (BR) multigene family members in the dipteran Chironomus tentans encode salivary gland secretory proteins in the size range between 38 and 1,000 kDa. The proteins interact to form protein fibers used by the aquatic larvae to spin feeding and protective larval tubes or pupation tubes. Here, we describe a new BR multigene family member, the spl7 gene, which codes for an 89-amino-acid-long protein with a relative mobility of 17k. The gene has a high content of charged amino acid residues and consists of two structurally different halves. Five regularly spaced cysteine codons are present in the 5′ half while the 3′ half contains five proline codons. These two different halves exhibit similarities to the C and SR regions, respectively, which form the tandemly repeated units in the about 40-kb-long BR genes and which also, in different versions, are the building blocks of all genes in the BR multigene family.In this multigene family, encoding interacting structural proteins, the long BR genes with their 125–150 tandemly arranged repeat units as well as the short sp17 gene with its single-copy version of such a repeat unit, have therefore evolved from a common ancestor.