Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection. Received: 7 March 1999 / Accepted: 25 March 1999     

Genomic organization and chromosomal mapping of mouse nuclear factor kappa B 2 (NFKB2)

 NFKB2 is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase and immune responses. NF-κB2 is initially synthesized as a ∼100 000 M _r protein which needs to be processed in order to bind DNA, either as homodimer or as heterodimer with other members of the NF-κB/Rel family. The unprocessed form of NF-κB2 acts as an IκB-like protein. Therefore, NF-κB2 has a dual function. In this report we describe the genomic structure, expression pattern, and chromosomal localization of mouse NFKB2. Genomic clones were isolated, which span the entire gene of approximately 8.5 kilobases (kb) including 1.5 kb of the promoter region. Comparison to its human and avian homologues revealed a strong evolutionary conservation of the gene structure including the exon/intron borders, sequence, and position of the nuclear localization signal, the glycine-hinge region, and the ankyrin repeats. By fluorescence in situ hybridization, mouse NFKB2 was mapped to Chromosome (Chr) MMU 19C3-D2, which is homologous to human Chr 10q24, at which position the human NFKB2 was previously located. NFKB2 is ubiquitously expressed, highest in lymph nodes and thymus, underlining its role in the immune function. Received: 14 January 1999 / Revised: 29 March 1999     

The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30–45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented. Received: 25 November 1999 / Revised: 10 January 2000     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part IV)

Summary.  Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the molecular mechanisms of the various phenotypes of DS could be due to overexpression of gene(s) on chromosome 21, several groups have challenged this gene dosage effect hypothesis. The near completion of the sequencing of human chromosome 21 provides unprecedented opportunities to understand the molecular pathology of DS, however, functional information on gene products is limited so far. We therefore evaluated the levels of six proteins whose genes are encoded on chromosome 21 (trefoil factor 1, trefoil factor 2, trefoil factor 3, coxsackie virus and adenovirus receptor, carbonyl reductase 1 and interferon-α receptor) in fetal cerebral cortex from DS and controls at the early second trimester using Western blot analysis. None of the investigated proteins showed overexpression in DS compared to controls suggesting that these proteins are not involved in abnormal development of fetal DS brain and that DS phenotype can not be simply explained by the gene dosage effect hypothesis. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 and these studies may provide a better understanding of genotype-phenotype correlation in DS. Received November 28, 2002 Accepted March 10, 2003 Acknowledgements's of Hospital of Philadelphia, PA, (USA) and Biogen, Inc. (anti-IFNAR-1 antibody; Cambridge, USA) for kindly providing the antibodies and comments. Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AD, Alzheimer's disease; CAR, coxsackievirus and adenovirus receptor; CBR1, carbonyl reductase 1; CNS, central nervous system; DS, Down syndrome; IFNs, interferons; IFNAR-1, interferon-α receptor; NSE, neuron specific enolase; TFF, trefoil factor     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II)

Summary.  Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, αA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. Received July 1, 2002 Accepted July 19, 2002 Published online November 14, 2002 Acknowledgement This work was supported, in part (Dr. D. Patterson), by the National Institute of Child Health and Human Development (NICHD; HD17449). Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: DS, Down syndrome; HACS1, hematopoietic adapter containing Src homology 3 domain and sterile α motifs; DYRK1A, dual specificity tyrosine phosphorylated and regulated kinase; αA-crystallin, alpha crystallin subunit A; FTCD, formi-minotransferase cyclodeaminase; GARS-AIRS-GART, glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase; CBS, cystathionine β-synthase; NSE, neuron specific enolase; GFAP, glial fibrillary acidic protein     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I)

Summary.  Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this “gene dosage effect” hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18–19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis. Received June 27, 2002 Accepted July 19, 2002 Published online November 14, 2002 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AIRE, autoimmune regulator; DS, Down syndrome; HCS, holocarboxylase synthetase; Prep1, Pbx regulating protein 1; Tiam1, T-cell lymphoma invasion and metastasis 1     

Genomic organization of the extracellular coding region of the human FGFR4 and FLT4 genes: Evolution of the genes encoding receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases with five, seven, and three Ig-like domains in their extracellular region are grouped in subclasses IIIa, IIIb, and IIIc, respectively. Here, we describe the genomic organization of the extracellular coding region of the human FGFR4 (IIIc) and FLT4 (IIIb) genes and compare it to that of the human FGFR1(IIIc), KIT, and FMS (IIIa). The results show that while genes belonging to the same subclass have an identical exon/intron structure in their extracellular coding region—as they do in their intracellular coding region—genes of related subclasses only have a similar exon/intron structure. These results strongly support the hypothesis that the genes of the three subclasses evolved from a common ancestor by duplications involving entire genes, already in pieces. Hypotheses on the origin of introns and on the difference in the number of extracellular Ig-like domains in the three gene subclasses are discussed. Received: 19 August 1996 / Accepted: 2 January 1997     

Allelic diversity at the Mhc-DQA locus in cotton rats (Sigmodon hispidus) and a comparison of DQA sequences within the family Muridae (Mammalia: Rodentia)

 The cotton rat (Sigmodon hispidus) is a common murid rodent of the southern United States, Mexico, and Central America. Using single-stranded conformation polymorphism analysis and DNA sequencing techniques, 11 DQA exon 2 alleles were detected among 180 S. hispidus from Caddo County, Oklahoma, USA. The alleles represent a single locus exhibiting a high level of polymorphism. Nucleotide and amino acid distance values among DQA alleles of S. hispidus were higher than those within Mus musculus and species of Rattus. Although the distribution of polymorphic amino acid residues among alleles of S. hispidus was similiar to that of Mus and Rattus, some residues of the α-helix region were more variable in S. hispidus. Comparisons of nonsynonymous and synonymous substitutions indicated a trend toward higher numbers of nonsynonymous substitutions; however, this difference was not significant statistically among S. hispidus alleles. To examine evolution of DQA alleleswithin Muridae, we performed a phylogenetic analysis that included DQA alleles from S. hispidus, Peromyscus leucopus, M. musculus, R. norvegicus, and six Australian species of Rattus. Results depicted monophyly for each genus, and this concordance between species and gene trees represents a lack of evidence for trans-species persistence of alleles among these genera. Received: 22 October 1998 / Revised: 17 March 1999     

Coevolution of PERB11 (MIC) and HLA Class I Genes with HERV-16 and Retroelements by Extended Genomic Duplication

The recent availability of genomic sequence information for the class I region of the MHC has provided an opportunity to examine the genomic organization of HLA class I (HLAcI) and PERB11/MIC genes with a view to explaining their evolution from the perspective of extended genomic duplications rather than by simple gene duplications and/or gene conversion events. Analysis of genomic sequence from two regions of the MHC (the alpha- and beta-blocks) revealed that at least 6 PERB11 and 14 HLAcI genes, pseudogenes, and gene fragments are contained within extended duplicated segments. Each segment was searched for the presence of shared (paralogous) retroelements by RepeatMasker in order to use them as markers of evolution, genetic rearrangements, and evidence of segmental duplications. Shared Alu elements and other retroelements allowed the duplicated segments to be classified into five distinct groups (A to E) that could be further distilled down to an ancient preduplication segment containing a HLA and PERB11 gene, an endogenous retrovirus (HERV-16), and distinctive retroelements. The breakpoints within and between the different HLAcI segments were found mainly within the PERB11 and HLA genes, HERV-16, and other retroelements, suggesting that the latter have played a major role in duplication and indel events leading to the present organization of PERB11 and HLAcI genes. On the basis of the features contained within the segments, a coevolutionary model premised on tandem duplication of single and multipartite genomic segments is proposed. The model is used to explain the origins and genomic organization of retroelements, HERV-16, DNA transposons, PERB11, and HLAcI genes as distinct segmental combinations within the alpha- and beta-blocks of the human MHC. Received: 5 December 1998 / Accepted: 27 January 1999     

Genomic relationships within Boronia (Rutaceae) as revealed by karyotype analysis and RAPD molecular markers

 The genus Boronia Sm. section Boronia series Boronia contains species with n=7 (B. megastigma), n=7 or 8 (B. heterophylla), n=8 (B. molloyae) and n=9 (B. purdieana), representing ideal species with which to examine comparative chromosome morphology. Between species there were few chromosomes with similar morphology, indicating numerous genome re-organisations. Karyotypes between and within species of Boronia could be distinguished and inheritance of some chromosomes was observed. Species and hybrids with 2n = 14 or 15 had at least one large chromosome. Chromosome morphology indicated a closer relationship between B. heterophylla and B. molloyae and between B. purdieana and B. megastigma than between these two groups. Whole genomic DNA was extracted from 9 genotypes of Boronia. RAPD bands were analysed and pairwise distance matrices between genotypes were computed. Dendrograms were generated and analysed using unweighted pair-group method with arithmetic average cluster analysis. Dendograms supported cytological results, indicating B. heterophylla and B. molloyae are closely related and clearly distinct from B. megastigma and B. purdieana. The evolution of boronias is discussed. Received February 16, 2001; accepted March 21, 2002 Published online: October 14, 2002 Address of the authors: G. Yan, F. Shan, J. A. Plummer (e-mail: jplummer@cyllene.uwa.edu.au), Plant Sciences, Faculty of Natural and Agricultural Science, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.     

Duplication and Diversification of the Apolipoprotein CI (APOCI) Genomic Segment in Association with Retroelements

We have previously shown that several multicopy gene families within the major histocompatibility complex (MHC) arose from a process of segmental duplication. It has also been observed that retroelements play a role in generating diversity within these duplicated segments. The objective of this study was to compare the genomic organization of a gene duplication within another multicopy gene family outside the MHC. Using new continuous genomic sequence encompassing the APOE-CII gene cluster, we show that APOCI and its pseudogene, APOCI′, are contained within large duplicated segments which include sequences from the hepatic control region (HCR). Flanking Alu sequences are observed at both ends of the duplicated unit, suggesting a possible role in the integration of these segments. As observed previously within the MHC, the major differences between the segments are the insertion of sequences (approximately 200–1000 bp in length), consisting predominantly of Alu sequences. Ancestral retroelements also contribute to the generation of sequence diversity between the segments, especially within the 3′ poly(A) tract of Alu sequences. The exonic and regulatory sequences of the APOCI and HCR loci show limited sequence diversity, with exon 3 being an exception. Finally, the typing of pre- and postduplication Alus from both segments indicates an estimated time of duplication of approximately 37 million years ago (mya), some time prior to the separation of Old and New World monkeys. Received: 17 July 1999 / Accepted: 6 November 1999     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part III)

Summary.  Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS. Received August 12, 2002 Accepted September 12, 2002 Published online January 30, 2003 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK) Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: ADAR2, adenosine deaminase RNA-specific 2; C21orf2, chromosome 21 open reading frame 2; DS, Down syndrome; NSE, neuron specific enolase; OSCP, oligomycin sensitivity conferring protein; PEP-19, peptide 19     

Isolation and mapping of the rabbit DM genes

 Proper peptide presentation by major histocompatibility complex (MHC)-encoded class II antigens is dependent on the products of the MHC DM loci. We identified the rabbit orthologues (RLA-DMA and -DMB) of human HLA-DMA and -DMB and found that they have 76.9% and 78.8% identity with HLA-DMA and -DMB, respectively. Like classical class II MHC genes, RLA-DM genes are more closely related to human HLA-DM genes than to mouse H2-DM. Among the DM family, there is a high degree of variability at the amino terminus of the DMa chains, and length variability in the cytoplasmic tails of both DMα and DMβ. The rabbit DM genes are coexpressed with class II genes in lymphoid tissues, as are the DM genes of other mammals. The RLA-DM locus maps to the class II region of the rabbit MHC, and is flanked by the DP and DOB loci. Despite having some similarities to class II genes of bony fishes, the DM family represents a separate branch of the MHC class II family. Received: 30 May 1998 / Revised: 28 July 1998     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998