A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

High-resolution physical mapping by combined Alu-hybridization/PCR screening: construction of a yeast artificial chromosome map covering 31 centimorgans in 3p21-p14.

We describe an integrated approach to large-scale physical mapping using an Alu-PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands p14-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of approximately equal to 100 kb. We observe an inverse distribution of Alu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high-resolution human physical maps.

Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus.

The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Atomic force microscope investigation of large-circle DNA molecules

A circular bacterial artificial chromosome of 148.9 kbp on human chromosome 3 has been extended and fixed on bare mica substrates using a developed fluid capillary flow method in evaporating liquid drops. Extended circular DNA molecules were imaged with an atomic force microscope (AFM) under ambient conditions. The measured total lengths of the whole DNA molecules were in agreement with sequencing analysis data with an error range of +/-3.6%. This work is important groundwork for probing single nucleotide polymorphisms in the human genome, mapping genomic DNA, manipulating biomolecular nanotechnology, and studying the interaction of DNA-protein complexes investigated by AFM.

Identificação de genes MUTM em BACs da cana-de-açucar e caracterização preliminar destes genes e seus promotores

Sugarcane has an importance in Brazil due to sugar and biofuel production. Considering this aspect, there is basic research being done in order to understand its physiology to improve production. The aim of this research is the Base Excision Repair pathway, in special the enzyme MUTM DNA-glycosylase (formamidopyrimidine) which recognizes oxidized guanine in DNA. The sugarcane scMUTM genes were analyzed using four BACs (Bacterial Artificial Chromosome) from a sugarcane genomic library from R570 cultivar. The resulted showed the presence in the region that had homology to scMUTM the presence of transposable elements. Comparing the similarity, it was observed a highest similarity to Sorghum bicolor sequence, both nucleotide and peptide sequences. Furthermore, promoter regions from MUTM genes in some grass showed different cis-regulatory elements, among which, most were related to oxidative stress, suggesting a gene regulation by oxidative stress

Identification and Analyses of Candidate Genes for Rpp4-Mediated Resistance to Asian Soybean Rust in Soybean

Asian soybean rust is a formidable threat to soybean (Glycine max) production in many areas of the world, including the United States. Only five sources of resistance have been identified (Resistance to Phakopsora pachyrhizi1 [Rpp1], Rpp2, Rpp3, Rpp4, and Rpp5). Rpp4 was previously identified in the resistant genotype PI459025B and mapped within 2 centimorgans of Satt288 on soybean chromosome 18 (linkage group G). Using simple sequence repeat markers, we developed a bacterial artificial chromosome contig for the Rpp4 locus in the susceptible cv Williams82 (Wm82). Sequencing within this region identified three Rpp4 candidate disease resistance genes (Rpp4C1-Rpp4C3 [Wm82]) with greatest similarity to the lettuce (Lactuca sativa) RGC2 family of coiled coil-nucleotide binding site-leucine rich repeat disease resistance genes. Constructs containing regions of the Wm82 Rpp4 candidate genes were used for virus-induced gene silencing experiments to silence resistance in PI459025B, confirming that orthologous genes confer resistance. Using primers developed from conserved sequences in the Wm82 Rpp4 candidate genes, we identified five Rpp4 candidate genes (Rpp4C1-Rpp4C5 [PI459025B]) from the resistant genotype. Additional markers developed from the Wm82 Rpp4 bacterial artificial chromosome contig further defined the region containing Rpp4 and eliminated Rpp4C1 (PI459025B) and Rpp4C3 (PI459025B) as candidate genes. Sequencing of reverse transcription-polymerase chain reaction products revealed that Rpp4C4 (PI459025B) was highly expressed in the resistant genotype, while expression of the other candidate genes was nearly undetectable. These data support Rpp4C4 (PI459025B) as the single candidate gene for Rpp4-mediated resistance to Asian soybean rust.

A high-resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA

The major histocompatibility complex (MHC) in mammals codes for antigen-presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene-dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle-derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH5000), we generated a high-resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR5000) was aligned with the cattle MHC RH map (cR 12000) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two-point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single- and double-color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Building the sugarcane genome for biotechnology and identifying evolutionary trends

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Deletions of 11q22.3-q25 are associated with atypical lung carcinoids and poor clinical outcome

Carcinoids are slow-growing neuroendocrine tumors that, in the lung, can be subclassified as typical (TC) or atypical (AC). To identify genetic alterations that improve the prediction of prognosis, we investigated 34 carcinoid tumors of the lung (18 TCs, 15 ACs, and 1 unclassified) by using array comparative genomic hybridization (array CGH) on 3700 genomic bacterial artificial chromosome arrays (resolution ?1 Mb). When comparing ACs with TCs, the data revealed: i) a significant difference in the average number of chromosome arms altered (9.6 versus 4.2, respectively; P = 0.036), with one subgroup of five ACs having more than 15 chromosome arms altered; ii) chromosomal changes in 30% of ACs or more with additions at 9q (?1 Mb) and losses at 1p, 2q, 10q, and 11q; and iii) 11q deletions in 8 of 15 ACs versus 1 of 18 TCs (P = 0.004), which was confirmed via fluorescence in situ hybridization. The four critical regions of interest in 45% ACs or more comprised 11q14.1, 11q22.1-q22.3, 11q22.3-q23.2, and 11q24.2-q25, all telomeric of MEN1 at 11q13. Results were correlated with patient clinical data and long-term follow-up. Thus, there is a strong association of 11q22.3-q25 loss with poorer prognosis, alone or in combination with absence of 9q34.11 alterations (P = 0.0022 and P = 0.00026, respectively).

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Depletion of regulatory T cells augments a vaccine-induced T effector cell response against the liver-stage of malaria but fails to increase memory

Regulatory T cells (T(reg)) have been shown to restrict vaccine-induced T cell responses in different experimental models. In these studies CD4(+)CD25(+) T(reg) were depleted using monoclonal antibodies against CD25, which might also interfere with CD25 on non-regulatory T cell populations and would have no effect on Foxp3(+)CD25(-) T(reg). To obtain more insights in the specific function of T(reg) during vaccination we used mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin (DT) receptor-eGFP fusion protein under the control of the foxp3 gene locus (depletion of regulatory T cell mice; DEREG). As an experimental vaccine-carrier recombinant Bordetella adenylate cyclase toxoid fused with a MHC-class I-restricted epitope of the circumsporozoite protein (ACT-CSP) of Plasmodium berghei (Pb) was used. ACT-CSP was shown by us previously to introduce the CD8+ epitope of Pb-CSP into the MHC class I presentation pathway of professional antigen-presenting cells (APC). Using this system we demonstrate here that the number of CSP-specific T cells increases when T(reg) are depleted during prime but also during boost immunization. Importantly, despite this increase of T effector cells no difference in the number of antigen-specific memory cells was observed.

A novel amplicon at 8p22–23 results in overexpression of cathepsin B in esophageal adenocarcinoma

Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22–23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI–HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22–23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22–23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.

Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin

Coelomocytes, the heterogeneous population of sea urchin putative immune cells, were found to express a complex set of transcripts featuring scavenger receptor cysteine-rich (SRCR) repeats. SRCR domains define a metazoan superfamily of proteins, many of which are implicated in development and regulation of the immune system of vertebrates. Coelomocytes transcribe multiple SRCR genes from among a multigene family encoding an estimated number of 1,200 SRCR domains in specific patterns particular to each individual. Transcription levels for given SRCR genes may range from pronounced to undetectable, yet all tested animals harbor the genomic loci encoding these genes. Analysis of several SRCR genes revealed multiple loci corresponding to each type. In the case of one SRCR type, a cluster of at least three genes was detected within a 133-kb bacterial artificial chromosome insert, and conserved as well as unique regions were identified in sequences of three genomic clones derived from a single animal. Array hybridizations with repeated samples of coelomocyte messages revealed substantial alterations in levels of expression of many SRCR genes, with fluctuations of up to 10-fold in 1 week and up to 30-fold over a period of 3 months. This report is the first demonstration of genomic and transcriptional complexity in molecules expressed by invertebrate coelomocytes. The mechanisms controlling SRCR gene expression and the functional significance of this dynamic system await elucidation.