Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Construction of a bacterial artificial chromosome library of Endoclita excrescens as a tool for comparative gene mapping in Lepidoptera

Karyotype evolution in one of the most diverse and species‐rich group of insects, moths and butterflies (Lepidoptera), has interesting features that remain to be resolved. Recent studies showed that fluorescence in situ hybridization using bacterial artificial chromosome clones (BAC‐FISH) is an efficient cytogenetic method for identification and gene mapping of lepidopteran chromosomes. Using comparative mapping by BAC‐FISH, extensive synteny of genes was revealed between chromosomes of different lepidopteran species based on Bombyx mori genomic information. However, this comparative mapping has been done only in representatives of advanced groups of Lepidoptera. Here we constructed a BAC library of Endoclita excrescens, which belongs to the primitive lepidopteran family Hepialidae. High molecular weight DNA for the library construction was prepared from the pupae by using a rapid nuclear isolation method known in plants. The BAC clones of E. excrescens contain 66.6 kb inserts on average. The successful application of BAC‐FISH showed that the BAC library of E. excrescens is a useful tool for comparative gene mapping on chromosomes of this species.     

Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.     

Prenatal diagnosis and molecular cytogenetic characterization of a de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14)

We present prenatal diagnosis of de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14) and molecular cytogenetic characterization of the deletion using uncultured amniocytes. We review the phenotypic abnormalities of previously reported patients with similar proximal interstitial 4p deletions, and we discuss the functions of the genes of RBPJ, CCKAR, STIM2, PCDH7 and ARAP2 that are deleted within this region.     

Prenatal diagnosis of ring chromosome 2 with lissencephaly and 2p25.3 and 2q37.3 microdeletions detected using array comparative genomic hybridization

We present rapid aneuploidy diagnosis of ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions by aCGH using uncultured amniocytes in a fetus with IUGR, microcephaly, lissencephaly and ambiguous external genitalia. Our case adds lissencephaly to the list of CNS abnormalities in ring chromosome 2 with 2p25.3 and 2q37.3 microdeletions. We discuss the consequence of haploinsufficiency of HDAC4, KIF1A, PASK, HDLBP, FRAP2 and D2HGDH on 2q37.3, and haploinsufficiency of MYT1L, SNTG2 and TPO on 2p25.3 in this case.     

A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome

Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.     

Mosaic down syndrome with a marker: molecular cytogenetic characterization of the marker chromosome

Down syndrome is a complex disorder characterized by well defined and distinctive phenotypic features. Approximately 2-3% of all live-born Down individuals are mosaics. Here we report a boy with suspected Down syndrome showing mosaicism for two different cell lines where one cell line is unexpected. The cytogenetic analysis by G-banding revealed a karyotype of 47 XY+21 [20]/46,X+marker [30]. Further, molecular cytogenetic analysis with spectral karyotyping identified the marker as a derivative of Y chromosome. The delineation of Y chromosomal DNA was done by quantitative real-time PCR and aneuploidy detection by quantitative fluorescence PCR. The Y-short tandem repeats typing was performed to estimate the variation in quantity as well as to find out the extent of deletion on Y chromosome using STR markers. Fluorescence in situ hybridization using Y centromeric probe was also performed to confirm the origin of the Y marker. Further fine mapping of the marker was carried out with three bacterial artificial chromosome clones RP11-20H21, RP11-375P13, RP11-71M14, which defined the hypothetical position of the deletion. In our study we defined the extent of deletion of the marker chromosome and also discussed it in relation with mosaicism. This is the first report of mosaic Down syndrome combined with a second de novo mosaic marker derived from the Y chromosome.     

Prenatal diagnosis and molecular cytogenetic characterization of de novo partial trisomy 12q (12q24.21 → qter) and partial monosomy 6q (6q27 → qter) associated with coarctation of the aorta,ventriculomegaly and thickened nuchal fold

We present rapid aneuploidy diagnosis of de novo partial trisomy 12q (12q24.21 → qter) and partial monosomy 6q (6q27 → qter) by aCGH using uncultured amniocytes in a fetus with coarctation of the aorta, ventriculomegaly and thickened nuchal fold. We discuss the association of TBX3, TBX5 and MED13L gene duplication with coarctation of the aorta, and the association of RNASET2 gene haploinsufficiency with ventriculomegaly in this case.     

A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip

Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co‐localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3‐CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α‐ketoglutarate‐dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p‐coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.     

Characterization of a novel nm23 gene and its potential roles in gametogenesis in the prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea: Decapoda)

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776 bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH₂-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

A swimy locus on Y chromosome of the platyfish (Xiphophorus maculatus) is derived from a novel DNA transposon Zisupton

A swimy locus derived from a novel DNA transposon Zisupton was located on the sex determination region (SD) of Xiphophorus maculatus. The analysis of expression pattern showed that swimy was exclusively expressed in adult testis in X. maculatus. The putative 939 aa sequence contains four Zn-finger domains, such as two C2H2 type, one NFX type and one SWIM type Zn-finger domain, and one SAP DNA-binding domain. Swimy has about 7 copies per haploid X. maculatus genome with Y-specific copies located in the SD region, and become the second new W-linked marker of platyfish. Analysis of the structure and distribution of this sex-linked marker is benefit to shed new light on the evolutionary dynamics of sex chromosomes in fish.     

Identification of a novel missense mutation of PEX7 gene in an Iranian patient with rhizomelic chondrodysplasia punctata type 1

Deficiency in the PTS2 protein import pathway due to mutations in PEX7 gene results in the rhizomelic chondrodysplasia punctata (RCDP) type 1. In the present study, we have reported a novel missense mutation, W75R, in the PEX7 gene in an Iranian patient with the RCDP type 1. The inability of PEX7 protein to transport PTS2 containing proteins including peroxisomal 3-ketoacyl-CoA thiolase and PTS2-EGFP protein to the surface of the peroxisomes showed that the W75R mutation in PEX7 gene severely impaired the function of PEX7 protein and was responsible for RCDP type 1 in this patient.     

Mapping breakpoints of a familial chromosome insertion (18,7) (q22.1; q36.2q21.11) to DPP6 and CACNA2D1 genes in an azoospermic male

It is widely accepted that the incidence of chromosomal aberration is 10–15.2% in the azoospermic male; however, the exact genetic damages are currently unknown for more than 40% of azoospermia. To elucidate the causative gene defects, we used the next generation sequencing (NGS) to map the breakpoints of a chromosome insertion from an azoospermic male who carries a balanced, maternally inherited karyotype 46, XY, inv ins (18,7) (q22.1; q36.2q21.11). The analysis revealed that the breakage in chromosome 7 disrupts two genes, dipeptidyl aminopeptidase-like protein 6 (DPP6) and contactin-associated protein-like 2 (CACNA2D1), the former participates in regulation of voltage-gated potassium channels, and the latter is one of the components in voltage-gated calcium channels. The deletion and duplication were not identified equal or beyond 100 kb, but 4 homologous DNA elements were verified proximal to the breakpoints. One of the proband's sisters inherited the same aberrant karyotype and experienced recurrent miscarriages and consecutive fetus death, while in contrast, another sister with a normal karyotype experienced normal labor and gave birth to healthy babies. The insertional translocation is confirmed with FISH and the Y-chromosome microdeletions were excluded by genetic testing. This is the first report describing chromosome insertion inv ins (18,7) and attributes DPP6 and CACNA2D1 to azoospermia.     

Identification and characterization of a differentially expressed protein (CAPZB) in skeletal muscle between Meishan and Large White pigs

Actin capping protein beta (CAPZB) protein was identified with considerable differences in the longissimus dorsi muscle between Large White and Meishan pigs using proteomics approach. However, in pigs, the information on CAPZB is very limited. In this study, we cloned and characterized the porcine actin capping protein beta (CAPZB) gene. In addition, we present two novel porcine CAPZB splice variants CAPZB1 and CAPZB2. CAPZB1 was expressed in all twenty tissues. However, CAPZB2 was predominantly expressed in the skeletal muscle and heart. In addition, the two isoforms had different expression profiles during the skeletal muscle development and between breeds. Moreover, the SNP T394G was identified in the coding region of the CAPZB gene, which was significantly associated with the carcass traits including the LFW, CFW, SFT and LEA. Data presented in our study suggests that the CAPZB gene may be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle.     

A particular set of small non-coding RNAs is bound to the distinctive Argonaute protein of Trypanosoma cruzi: Insights from RNA-interference deficient organisms

The study of small RNAs and Argonaute proteins in eukaryotes that are deficient in functional RNA interference could provide insights into novel functions of small RNAs. In this study we describe small non-coding RNAs bound to a distinctive Argonaute protein of Trypanosoma cruzi, TcPIWI-tryp. Co-immunoprecipitation of TcPIWI-tryp followed by deep sequencing of isolated RNA identified abundant small RNAs derived from rRNAs and tRNAs. The small RNA repertoire differed from that of the canonical Argonaute in organisms with functional RNA interference, which could indicate novel biological functions for TcPIWI-tryp in T. cruzi and other members of the trypanosomatid clade.