Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Identification and disruption of the gene encoding the third member of the low-molecular-mass rhoptry complex in Plasmodium falciparum

The low-molecular-mass rhoptry complex of Plasmodium falciparum consists of three proteins, rhoptry-associated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the RAP3 gene has not been identified. In this study we identify the RAP3 gene from the P. falciparum genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of RAP3 demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all Plasmodium spp., suggesting that this protein plays a role in merozoite invasion. The region surrounding the RAP3 homologue in the Plasmodium yoelii genome is syntenic with the same region in P. falciparum; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from Plasmodium spp. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.

Development of Y-chromosome-Specific SCAR Markers Conserved in Taurine, Zebu and Bubaline Cattle

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

Origin and differentiation of a sex chromosome system in Parodon hilarii (Pisces, Parodontidae). Satellite DNA, G- and C-banding

A satellite DNA sequence of Parodon hilarii ( named pPh2004) was isolated, cloned and sequenced. This satellite DNA is composed of 200 bp, 60% AT rich. In situ hybridization ( FISH) results revealed that the satellite DNA pPh2004 is located in the terminal regions of several chromosomes, forming highly evident blocks in some and punctual marks in others. The comparison between the FISH and C-banding results showed that the location of this satellite DNA coincides with that of most terminal heterochromatins. However, some regions are only marked by FISH whereas other regions are only marked by C-banding. The possible existence of more than one satellite DNA family could explain these partial differences. The in situ hybridization with the satellite DNA and the G- and C-bandings confirmed the presence of a sex chromosome system of the ZZ/ZW type in P. hilarii, as well as the correct identification of the Z chromosome in the karyotype. This chromosome displays a segment of terminal heterochromatin in the long arm, similar to the segment observed in the short arm of the W chromosome, also showing a G- banding pattern similar to that of the short arm and part of the long arm of the W chromosome. A hypothesis on the origin of the W chromosome from an ancestral chromosome similar to the Z chromosome is presented.

X-chromosome genetic variation in São Paulo State (Brazil) population

As data on X-chromosomal short tandem repeats (X-STRs) for the Brazilian population are scarse, the aim of this study was to determine the allele frequencies of five X-STRs (DXS6854, DXS7424, DXS101, DXS6808 and DXS7132) in the São Paulo State, Brazil. No deviations from the Hardy-Weinberg equilibrium were observed, with the exception of DXS101. The forensic efficiency parameters demonstrated that DXS101 was the most informative marker. Population comparisons revealed that the X-STR profile sampled in the state of São Paulo was more similar to European and African populations than Asiatic populations reported in this work.

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

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

Identification of a Microdeletion at the 7q33-q35 Disrupting the CNTNAP2 Gene in a Brazilian Stuttering Case

Speech and language disorders are some of the most common referral reasons to child development centers accounting for approximately 40% of cases. Stuttering is a disorder in which involuntary repetition, prolongation, or cessation of the sound precludes the flow of speech. About 5% of individuals in the general population have a stuttering problem, and about 80% of the affected children recover naturally. The causal factors of stuttering remain uncertain in most cases; studies suggest that genetic factors are responsible for 70% of the variance in liability for stuttering, whereas the remaining 30% is due to environmental effects supporting a complex cause of the disorder. The use of high-resolution genome wide array comparative genomic hybridization has proven to be a powerful strategy to narrow down candidate regions for complex disorders. We report on a case with a complex set of speech and language difficulties including stuttering who presented with a 10Mb deletion of chromosome region 7q33-35 causing the deletion of several genes and the disruption of CNTNAP2 by deleting the first three exons of the gene. CNTNAP2 is known to be involved in the cause of language and speech disorders and autism spectrum disorder and is in the same pathway as FOXP2, another important language gene, which makes it a candidate gene for causal studies speech and language disorders such as stuttering. (C) 2010 Wiley-Liss, Inc.

Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae)

Individuals of two populations of the fish Characidium cf. fasciatum were cytogenetically studied and showed a basic diploid number of 50 chromosomes. Some fishes were found to have 51 to 54 chromosomes due to the presence of one to four small subtelocentric/acrocentric supernumerary chromosomes. When analyzed by conventional Giemsa staining, male and female specimens of C. cf. fasciatum from the Quinta stream and Pardo River presented the same basic karyotypic macro- and microstructure, consisting of 32 metacentric and 18 submetacentric chromosomes.Ag-NORs were terminally located on the long arms of two submetacentric chromosome pairs. Constitutive heterochromatin was identified by C-banding as small pericentromeric blocks in the majority of the chromosomes, and B-chromosomes were found to be heterochromatic. The occurrence of one totally heterochromatic submetacentric chromosome restricted to females and considered as an unusual feature in fish karyotypes led to the identification of a ZZ/ZW sex-chromosome system. The implications of chromosomic differentiation observed in the genus Characidium are discussed.

Identification of a new repetitive element in the sex chromosomes of Leporinus elongatus (Teleostei : Characiformes : Anostomidae): new insights into the sex chromosomes of Leporinus

Leporinus elongatus represents an interesting model for studies on chromosome evolution since it possesses a conspicuous ZZ/ZW sex chromosome system that has been characterized mainly by basic cytogenetic techniques. In the present study we describe a dispersed repetitive element ( named Le SpeI) related to the sex chromosomes of L. elongatus. Females revealed clusters of Le SpeI on the long arm of the W chromosome and in the acrocentric NOR-bearing chromosome pair. In males, the signal was restricted to the pericentromeric region of the NOR-bearing chromosomes. Considering the results obtained in the present study using FISH, NOR and C-banding, together with findings from previous studies, it can be inferred that the sex chromosome system of L. elongatus is still undergoing an evolutionary process. The data suggest novelties in relation to the sex chromosomes of the genus Leporinus with the description of a multiple sex chromosome system involving the NOR-bearing chromosomes. Therefore, it is hypothesized that the simple ZW chromosome system previously described for L. elongatus rather is a multiple Z(1)Z(1)Z(2)Z(2)/Z(1)W(1)Z(2)W(2) system. Copyright (c) 2007 S. Karger AG, Basel

Identification and description of distinct B chromosomes in Cyphocharax modestus (Characiformes, Curimatidae)

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

Physical chromosome mapping of repetitive DNA sequences in Nile tilapia Oreochromis niloticus: Evidences for a differential distribution of repetitive elements in the sex chromosomes

Repetitive DNAs have been extensively applied as physical chromosome markers on comparative studies, identification of chromosome rearrangements and sex chromosomes, chromosome evolution analysis, and applied genetics. Here we report the characterization of repetitive DNA sequences from the Nile tilapia (Oreochromis niloticus) genome by construction and screening of plasmid library enriched with repetitive DNAs, analysis of a BAC-based physical map, and hybridization to chromosomes. The physical mapping of BACs enriched with repetitive sequences and C(o)t-1 DNA (DNA enriched for highly and moderately repetitive DNA sequences) to chromosomes using FISH showed a predominant distribution of repetitive elements in the centromeric and telomeric regions and along the entire length of the largest chromosome pair (X and Y sex chromosomes) of the species. The distribution of repetitive DNAs differed significantly between the p arm of X and Y chromosomes. These findings suggest that repetitive DNAs have had an important role in the differentiation of sex chromosomes. (c) 2007 Elsevier Ltd. All rights reserved.

Identification and characterization of different accessions of itchgrass (Rottboellia cochinchinensis)

Studies were conducted to identify and characterize different accessions of itchgrass. Seeds were collected in the counties of Aramina, Campinas, Dumont, Igarapava, Jaboticabal, and Ribeirao Preto, all in the state of São Paulo, Brazil. Accessions were characterized based on dimensions of their stomata, stomatal index (SI), and length and width of their seed (caryopses and husk). Chromosome number and length also were determined, and accessions were further differentiated using molecular markers (polymerase chain reaction [PCR]). Itchgrass from Ribeirao Preto had much longer and narrower seeds than those from the other locations, and their husks were longer as well. Accessions had similar SIs, both on the abaxial and adaxial leaf surfaces. Stomata from Campinas and Igarapava accessions were longer and wider, whereas those from Dumont and Ribeirao Preto were similar and smaller than all others. The accession from Ribeirao Preto is diploid (2n = 20); the rest are polyploid, with the total length of chromosomes smaller than all others. These differences were confirmed by molecular differentiation (PCR).

IDENTIFICATION OF COMMON FRAGILE SITES IN CHROMOSOMES OF 2 SPECIES OF BAT (CHIROPTERA, MAMMALIA)

In the karyotypes of the bat species Molossus ater and M molossus, spontaneous and bromodeoxyuridine (BrdU)- or aphidicolin (APC)-sensitive fragile sites were located. Four chromosome regions harbored APC-sensitive fragile sites: 1q9 and 8q4 in both M ater and M molossus, 3q3 in M ater, and 1p7 in M molossus. The fragile sites in 1q9 and 8q4 were also observed without induction in M molossus. BrdU-sensitive fragile sites were not detected. Despite observations in several other species, the fragile sites detected in Molossus are not coincident with the breakpoints involved in the chromosome rearrangements occurring in the evolution of 7 species of the Molossidae family.

Chromosome breakpoint distribution in nonmelanoma skin cancers

We have identified chromosome regions that may be sites of genes activated as a result of chromosomal rearrangements observed in 61 of the 86 skin tumors referenced in the literature. The data showed that most of the breakpoints were distributed throughout the genome and some tended to cluster. Highest frequencies of breakpoints were observed in chromosomes with high relative length, except chromosomes 14 and 15 that were more often affected in malignant tumors, despite their size. Our work provides a starting point for more detailed studies that may allow identification of these genes as important keys in the development and progression of skin cancers. (C) Elsevier B.V., 1997.

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.