Fourth Chromosome Research Articles

Comparative Genomics and Gene Annotation of a Region of Drosophila eugracilis Chromosome 4

The common fruit fly, Drosophilia melanogaster, has around 1,300 genes in its genome. Chromosome 4 is 3.5% of its genome and contains approximately 80 genes within large heterochromatic domains. Interestingly, these genes are expressed at similar levels to genes found in euchromatin. The goal of the Genomics Education Partnership (GEP) project is to understand the gene expression within this heterochromatic environment. For the GEP project, we are identifying conserved DNA regulatory sequences around transcription start sites using the fourth chromosome from other species of Drosophila and D. melanogaster. I annotated genes located within a 35,000 base‐pair section of Drosophilia eugracilis chromosome 4 using BLASTX, Gene predictors, and RNA‐seq evidence within the UCSC Genome Browser. I annotated coding exons and splice sites for 2 genes with 3 isoforms each, Cubitis interruptus (Ci) and Ribosomal protein S3A (RpS3A) and the transcription start sites for each gene. GEP students are annotating many megabases of DNA and their pooled results are furthering an understanding of heterochromatic gene structure and regulation.Support or Funding InformationSupported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Annotation of Genes on Contig 31 of the Drosophila eugracilis Chromosome 4

Drosophila melanogaster has a small fourth chromosome, the Muller F element (3.5% of the genome), that has very large heterochromatin domains which are rich in HP1. Our goal for this project is to understand the mechanisms of gene expression in this heterochromatic environment. In the Genomics Education Partnership Project, we are using comparative genomics to identify conserved DNA regulatory sequences, by comparing the D. melanogaster Muller F element to those of other fly species that diverged from D. melanogaster ~10 million years ago, aiming to find regions of homology surrounding the transcription start sites. I have annotated the genes on a 40,000 base‐pair piece from the Drosophila eugracilis chromosome four. Sequence comparisons using BLASTX, gene predictors like Gene Scans, and RNA sequence data were used to annotate coding genes in this region. I identified two genes: myo (3 isoforms) and ey (4 isoforms) and annotated protein coding exons and splice sites for each isoform. The myo amino acid sequence is identical for each isoform in D. eugracilis, with 70.3% amino acid identity compared to the D. melanogaster myo protein. The D. eugracilis ey isoforms are also similar to the D. melanogaster ey protein sequences, with a range from 73% to 78% identity to their corresponding D. melanogaster isoform. Putative Transcription Start Sites for each gene were investigated, including a search for known transcription factor binding sites. By pooling results, GEP students are annotating several megabases of DNA, and contributing to an understanding of heterochromatic genes.Support or Funding InformationSupported by NSF IUSE #1431407 and NIH R25GM119157 to Sarah C.R. Elgin (GEP) and NSF HRD#1623340 to MVSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leptin Gene as Potential Gene for Molecular Selection on Cattle in Indonesia

<p>Genetic improvement in beef and dairy cattle which can increase meat and milk production can be conducted through livestock selection based on molecular technique using leptin gene. The leptin gene is located on the fourth chromosome and consists of three exons and two introns. The leptin gene serves to produce the leptin hormone secreted by fat tissue. High concentrations of leptin hormone can increase feed intake and body metabolism thereby increasing livestock productivity. This paper describes the leptin gene including structure, mechanism of leptin hormone and polymorphisms in cows. These polymorphisms occur in the promoter, intron and exon sections associated with body and carcass weight and milk production in some cattle breed. Therefore, leptin gene can be used for selection towards increasing cattle productivity. Utilization of leptin gene can be done by single nucleotide polymorphism (SNP) identification on the whole structure of leptin gene and to observe its effect on characteristics of cattle production in Indonesia.</p>

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.

MYC gene family in cereals: Transformations during evolution of hexaploid bread wheat and its relatives

The transcription factors of the MYC gene family are an integral part of the MYB + MYC + WD40 regulatory complex required to activate the genes of plant flavonoid biosynthesis. The TaMyc1 gene, which controls the synthesis of flavonoid pigments in the grain pericarp, is known in bread wheat (Triticum aestivum L., BBAADD genome, 2n = 6x = 42). In the present work, we identified 10 copies of this gene in the T. aestivum genome, 22 copies in the nearest bread wheat relatives (T. durum, T. urartu, T. monococcum, Aegilops speltoides, Ae. sharonensis, Ae. tauschii). The analysis of genetic similarity of all these genes demonstrated that the MYC gene duplication occurred for the first time in the common diploid ancestor of the Triticeae tribe with the formation of copies in the second and fourth chromosomes. In the members of the Triticum and Aegilops genera, these genes underwent from two to four duplication acts that resulted in the formation of paralogous copies. The orthologs of the MYC genes obtained from ancestral diploid species exist in polyploid species of the Triticum genus (in addition to paralogues). The time of the emergence of individual MYC family members was calculated based on the average speed of accumulation of nucleotide substitutions (k) in the MYC genes (established in this work) and certain number of synonymous substitutions between individual copies.

Sex Differences in Drosophila Somatic Gene Expression: Variation and Regulation by doublesex

Sex differences in gene expression have been widely studied in Drosophila melanogaster. Sex differences vary across strains, but many molecular studies focus on only a single strain, or on genes that show sexually dimorphic expression in many strains. How extensive variability is and whether this variability occurs among genes regulated by sex determination hierarchy terminal transcription factors is unknown. To address these questions, we examine differences in sexually dimorphic gene expression between two strains in Drosophila adult head tissues. We also examine gene expression in doublesex (dsx) mutant strains to determine which sex-differentially expressed genes are regulated by DSX, and the mode by which DSX regulates expression. We find substantial variation in sex-differential expression. The sets of genes with sexually dimorphic expression in each strain show little overlap. The prevalence of different DSX regulatory modes also varies between the two strains. Neither the patterns of DSX DNA occupancy, nor mode of DSX regulation explain why some genes show consistent sex-differential expression across strains. We find that the genes identified as regulated by DSX in this study are enriched with known sites of DSX DNA occupancy. Finally, we find that sex-differentially expressed genes and genes regulated by DSX are highly enriched on the fourth chromosome. These results provide insights into a more complete pool of potential DSX targets, as well as revealing the molecular flexibility of DSX regulation.

Karyomorphological study of five Turkish endemicRhaponticoidesVaill. (Asteraceae, Cardueae) species

AbstractIn this paper, we aimed to determine the chromosomal features of Turkish endemic Rhaponticoides species. The chromosome numbers and karyomorphology of five species were examined. The chromosomal counts confirmed the previous reports of 2n = 30 given for Rhaponticoides species growing in different countries . Although R. iconiensis had a karyotype formula of 28m + 2sm, the remaining four species all had 26m + 4sm. Rhaponticoides aytachii and R. hierroi were especially similar in asymmetry indices. Rhaponticoides iconiensis and R. phytiae had satellites in the fourth and second chromosome pairs, respectively. Having the most symmetrical chromosomes, R. amasiensis displayed an important relationship with R. iconiensis and R. phytiae in terms of total chromosome length (TCL). If we make an overall assessment in terms of chromosomal asymmetry, chromosomes indices continues in the low range for each species and this low asymmetry indeces indicate that the species have symmetric chromosomes as karyomorph...

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly.

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.

Using Comparative Genomics to Study DNA Sequences of the Muller F Element from Drosophila biarmipes

The Genomics Education Partnership* (GEP) is a scientific and educational partnership using comparative genomics to characterize the Muller F element, a distinct chromosomal domain, from the genomes of several Drosophila species (see www.gep.wustl.edu). In Drosophila melanogaster, the F element corresponds to the fourth chromosome (or “dot” chromosome), which appears to be heterochromatic by many criteria, but has a relatively high gene density. Our goal is to study the evolution of these sequences to further our understanding of the function of heterochromatin. As part of an advanced genetics course at Albion College, we used several web-based tools to annotate a 50,000 base pair contiguous sequence from the genome of Drosophila biarmipes. The annotation process involved using the corresponding genomic region of the Drosophila melanogaster fourth chromosome as a reference sequence. Using BLAST we estimated the positions of each exon, then using a variety of evidence tracks from the UCSC genome browser, we precisely localized start codons, donor and acceptor splice sites, and stop codons for each isoform. Gene models were verified using the Gene Model Checker program and by generating final sequence alignments and dot plots using BLAST. Our analysis resulted in the annotation of five distinct genes in this contig. The genes ranged in complexity from 2-7 splice variants, with individual isoforms ranging in size from 4 to 12 exons each. Our analysis will be combined with data from other student members of the partnership to help answer questions about the organization and function of the Muller F element in several different species. *The Genomics Education Partnership is funded by HHMI (Professors grant #52007051 to SCR Elgin) and by Washington University in St. Louis (WUSTL).

A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila.

The development of clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technologies promises a quantum leap in genome engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila melanogaster are still restricted to a few genes, use variable experimental conditions, and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different locations in the genome, including a gene on the fourth chromosome, at comparably high efficiencies. Our data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months.

The effects of purifying selection on patterns of genetic differentiation between Drosophila melanogaster populations.

Using the data provided by the Drosophila Population Genomics Project, we investigate factors that affect the genetic differentiation between Rwandan and French populations of D. melanogaster. By examining within-population polymorphisms, we show that sites in long introns (especially those >2000 bp) have significantly lower π (nucleotide diversity) and more low-frequency variants (as measured by Tajima's D, minor allele frequencies, and prevalence of variants that are private to one of the two populations) than short introns, suggesting a positive relationship between intron length and selective constraint. A similar analysis of protein-coding polymorphisms shows that 0-fold (degenerate) sites in more conserved genes are under stronger purifying selection than those in less conserved genes. There is limited evidence that selection on codon bias has an effect on differentiation (as measured by FST) at 4-fold (degenerate) sites, and 4-fold sites and sites in 8–30 bp of short introns ⩽65 bp have comparable FST values. Consistent with the expected effect of purifying selection, sites in long introns and 0-fold sites in conserved genes are less differentiated than those in short introns and less conserved genes, respectively. Genes in non-crossover regions (for example, the fourth chromosome) have very high FST values at both 0-fold and 4-fold degenerate sites, which is probably because of the large reduction in within-population diversity caused by tight linkage between many selected sites. Our analyses also reveal subtle statistical properties of FST, which arise when information from multiple single nucleotide polymorphisms is combined and can lead to the masking of important signals of selection.

Genomic Annotation of Drosophila biarmipes Dot Chromosome Contig 19 (LB531)

Comparative analysis of the genome of Drosophila melanogaster with other Drosophila species at various evolutionary distances can reveal information about differences in euchromatic and heterochromatic gene structure, and how evolutionary processes have changed the expression of genes located in these regions. The goal of this project, performed in conjunction with the Genomics Education Partnership at Washington University, was to annotate all the major features of Drosophila biarmipes dot chromosome Contig 19 ‐ including genes, pseudogenes, and repetitive sequences ‐ to generate a high quality section of the genome of this species. Basic Local Alignment Search Tool (BLAST) was used along with Gene Record Finder to compare the protein sequence of D. melanogaster exons to the D. biarmipes contig in order to locate the placement of the homologous exons in the latter species. Gene predicting programs were used to examine regions of the genome not immediately shown to contain genes. This information was entered into Gene Model Checker to compile a comprehensive map of the contig. Contig 19 is on the fourth (dot) chromosome of Drosophila biarmipes and it contains three genes, Syt7, Rad23 and Zip102B. Both the Syt7 and Rad23 genes showed high sequence conservation with their orthologs in D. melanogaster and are present in the same orientation. The Zip102B gene showed evidence of greater sequence divergence. Consistent RNAseq data and agreement between gene prediction programs led to the conclusion that this sequence divergence was due to the accumulation of point mutations relative to a common ancestral species. The contig displayed a repetitive element percentage of 40.32 percent, which is consistent with a heterochromatic DNA classification. No pseudogenes were identified in this contig.Grant Funding Source: Supported by HHMI grant # 52005780 to SCR Elgin at Washington University (St. Louis).

Low bone density in achondroplasia

To the editor, A 39-year-old female achondroplastic patient was admitted to our outpatient clinic for her back and lower extremity pain. Her height and weight were 125 cm and 38 kg, respectively (Fig. 1). She had a body mass index of 24.3 kg/m. She had undergone leg lengthening on bilateral tibias when she was 12. On physical examination, she had bilateral paravertebral muscle spasm at her back. She also had painful hip internal rotation and knee flexion on both sides with bilateral positive Clarke’s sign. Her physical examination was otherwise noncontributory. X-ray examination revealed early degenerative changes, especially at the hip joints and strikingly osteopenia. On detailed questioning, it was understood that she had been advised to receive 300,000 IU vitamin D3 (the form of vitamin D available in our country) once a year and to exercise regularly for low bone density 2 years ago. The past two bone mineral density (BMD) measurements, performed by a single technician using dual-energy X-ray absorptiometry (DEXA) on a Lunar DPX-IQ scanner in our center, were analyzed, and a new BMD measurement was performed under similar conditions with the ones referred above (Table 1, Fig. 2). The laboratory tests was as follows: serum total calcium, 9.3 mg/dl (8.2–10.6); inorganic phosphate, 3.2 mg/dl (2.5–4.5); alkaline phosphatase (ALP), 47 U/l (30–126); type 1 collagen C terminal telopeptide (CTX), 0.253 (25–573); osteocalcin, 3.46 ng/ml (3.1–13.7); parathormone, 34.23 pg/ml (15–65), and vitamin D3, 20.1 ng/ml (>30). Risk factor examination for low bone density yielded no results. Low bone density was considered as a consequence of altered bone metabolism due to achondroplasia. She was prescribed 300,000 IU oral vitamin D3 and recommended to continue on her exercises. She was also included in a physical therapy program for her symptoms due to degenerative changes. Achondroplasia is the most common form of skeletal dysplasias. A spontan or inherited mutation in the fibroblast growth factor receptor 3 (FGFR3) gene on the fourth chromosome brings out the disease [1]. The primary defect is abnormal endochondral ossification [2]. It has a number of wellknown neuromusculoskeletal manifestations [3]. Recently, Arita et al. reported low bone density, diagnosed with spinal BMD analysis and panoramic radiographic measurements, in 5 of 11 achondroplastic patients for the first time in the literature [4]. To the best of our knowledge, our report is the second one referring the relationship between

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, we demonstrate that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, our findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase.

Targeting of Painting of fourth to roX1 and roX2 Proximal Sites Suggests Evolutionary Links Between Dosage Compensation and the Regulation of the fourth Chromosome in Drosophila melanogaster

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome, and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4th chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. Here we present an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode noncoding RNAs important for the correct targeting and spreading of the MSL-complex. We also found that the targeting of POF to PoX1 and PoX2 is largely dependent on roX expression and identified a high-affinity target region that ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin.

The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila

The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.

Heterochromatic silencing in Drosophila melanogaster

Heterochromatin formation serves both to help organize and package large eukaryotic genomes, and to silence many of the transposable elements (TEs) that are an abundant component of these genomes. A critical question is how the cell decides which domains should be packaged in this form. Our prior studies on the heterochromatic fourth chromosome in Drosophila melanogaster have implicated the transposable element 1360 as a target for heterochromatin formation. 1360 remnants are concentrated in heterochromatic domains. Using a P element landing-pad construct to transpose TEs into the genome, we find that a euchromatic site close to a heterochromatic mass can be targeted for ectopic heterochromatin formation, as shown both by deposition of HP1a (ChIP-qPCR) and by the variegated silencing of the associated hsp70-white reporter gene. This outcome is achieved using either 1360 (a DNA transposon remnant) or Invader4 (a retrotransposon remnant) as the target TE. The effect is dependent on piRNA sites within the TE. The piRNA system is most active in the developing oocyte and early embryo. We find that knock-down of piwi or aubergine in the female germ line results in over-expression of many, but not all, TEs, with associated loss of HP1a and H3K9me2 from these sites. Depletion of Piwi in the maternal germ line and early zygote leads to a suppression of position effect variegation (PEV) from β-gal reporters assayed in the adult, while knock-down of piwi in the somatic cells of the developing eye linage has no such effect on an hsp70-white reporter. In contrast, depletion of HP1a at any stage (maternal germ line, early embryo, somatic cell linage) will cause a suppression of PEV as assayed in the adult. We propose a model in which the assembly of heterochromatin at PEV reporters in the early zygote is dependent on the RNAi system as well as structural heterochromatin proteins, with failure producing an effect that persists in the mature animal. In contrast, maintenance of heterochromatin once set appears not to require RNAi (specifically Piwi), but does require the structural proteins, specifically HP1a. piRNA-directed heterochromatin formation appears to be a significant mechanism for down-regulating transcription from some (not all) TEs, and a significant mechanism for targeting the silencing of PEV reporters. Supported by NIH grant GM068388.

Decoding the Disease-Associated Proteins Encoded in the Human Chromosome 4

Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (~6.4% of the human genome) with 757 protein-coding genes. A number of marker genes for many diseases have been found in this chromosome, including genetic diseases (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune system, cancer and stem cell) related genes (e.g., oncogenes, growth factors). As a pilot study for the chromosome 4-centric human proteome project (Chr 4-HPP), we present here a systematic analysis of the disease association, protein isoforms, coding single nucleotide polymorphisms of these 757 protein-coding genes and their experimental evidence at the protein level. We also describe how the findings from the chromosome 4 project might be used to drive the biomarker discovery and validation study in disease-oriented projects, using the examples of secretomic and membrane proteomic approaches in cancer research. By integrating with cancer cell secretomes and several other existing databases in the public domain, we identified 141 chromosome 4-encoded proteins as cancer cell-secretable/shedable proteins. Additionally, we also identified 54 chromosome 4-encoded proteins that have been classified as cancer-associated proteins with successful selected or multiple reaction monitoring (SRM/MRM) assays developed. From literature annotation and topology analysis, 271 proteins were recognized as membrane proteins while 27.9% of the 757 proteins do not have any experimental evidence at the protein-level. In summary, the analysis revealed that the chromosome 4 is a rich resource for cancer-associated proteins for biomarker verification projects and for drug target discovery projects.

Nuclear genome size and cytotype analysis inAgave cupreataTrel. & Berger (Agavaceae)

This study is a cytogenetic characterization by karyotyping and a determination of the DNA content by flow cytometry of wild populations of Agave cupreata from the Guerrero State, Mexico. Three of the studied populations were diploids (2n = 2x =60) and one population had tetraploid (2n = 4x = 120), pentaploid (2n = 5x = 150) and hexaploid (2n = 6x = 180) plants. Diploid populations had three different structural cytotypes. One population showed polyploid cytotypes. A. cupreata showed a bimodal karyotype of 10 large + 50 small chromosomes in diploids; 20 large + 100 small chromosomes in tetraploids and 25 large + 125 small chromosomes in pentaploids. In diploids, they had secondary constriction in one pair of the large chromosomes and, in the fourth or fifth large homologous chromosome groups, in polyploid plants. The arm ratio, the proportion of different types of large and small chromosomes, the mean of genome length and the asymmetry index of karyotypes clearly varied among diploid and polyploid cytotyp...

BCR/ABL Fusion Gene On Constitutional Der(14;22) Roberstsonian Translocation in a Case of Chronic Myeloid Leukemia with Masked Philadelphia Chromosome

AbstractAbstract 4822Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of the BCR/ABL chimeric gene. The remaining 5–10% of CML cases exhibit a variant Ph translocation generally involving a third or even a fourth chromosome in addition to chromosome 9 and 22, potentially leading to masked Ph chromosome or reveal cryptic translocations that remains undetected under conventional cytogenetic analysis. These chromosome rearrangements can be disclosed by means of fluorescence in situhybridization (FISH) or polymerase chain reaction (PCR) procedures. A very few Ph positive CML cases were reported with constitutional robertsonian translocations, i.e. translocation between two acrocentric chromomosomes (13–15, 21–22), with breakpoints in the short arms, leading to a dicentric chromosome and thus to 45 instead of 46 chromosomes Case Report.42 year-old woman presenting with asthenia. Physical examination: Grade 1 splenomegaly. Peripheral blood count showed: hemoglobin concentration 117g/L, platelet count: 329×109/L and white blood cell count (WBC): 199×109/L. Peripheral blood smear: myelemia exhibiting 3% of myeloid blasts. Cytogenetic analysis by G-banding performed on bone marrow metaphase cells afforded the following karyotype: 45, XX, der(14;22)(q10;q10)c?, t(9;22;11)(q34;q11;q13) [20]. The analysis of the BCR-ABLfusion gene according to standard protocols detected the presence of the b3a2 isoform. FISH studies using dual color dual fusion probes in metaphases showed a 1F2G2R signal pattern. We detect a normal ABL signal on chromosome 9 and BCR signal on chromosome 22; the fusion signal was present on the der(14;22);extra-signals BCR and ABL with reduced intensities were present on der(11) and der(9) respectively: ish der(9)(ABLdim+), der(11)(BCRdim+), der(14;22)(BCR+,ABL+) [10]. FISH analysis on interphase nuclei (n=200) presented the same signal pattern. Nuc ish (ABL, BCRx3)(BCR con ABL x1) [200]. Chromosome analysis of bone marrow cells after six months of Imatinib therapy showed the following karyotype: 45, XX, der(14;22)(q10;q10)c [20] thus demonstrating complete cytogenetic remission and that der(14;22) is a robertsonian constitutional abnormality that could be inherited and thus necessitate a familial genetic councelling to inform about the familial risk of congenital malformations and miscarriage. Discussion.To explain the formation of variant chromosome Ph translocations one-step, two-step and multi-step mechanisms have been proposed. In our case complex translocations involving four chromosomes and the participation of two acrocentric chromosomes, led to the hypothesis of the presence of a constitutional or acquired Robertsonian translocation. Karyotype analysis six months after treatment confirmed the presence of a constitutional Robertsonian translocation. According to the FISH pattern, this variant Ph chromosome was formed in one step.The occurrence of Philadelphia positive CML in a patient with a constitutional Robertsonian translocation is probably coincidental. The role of constitutional chromosomes abnormalities in hematologic malignancies is well known in Down syndrome patients and in chromosome breakage syndromes such as Fanconi anemia. In the literature, only one case of CML patients with Robertsonian t(14;22) have been described.To our knowledge this is the first report showing a Robertsonian t(14;22) in a variant Ph involving four chromosomes and exhibiting the fusion FISH signal in a derivative chromosome 14, with masked Ph. Disclosures:No relevant conflicts of interest to declare.