Mammalian adenylyl cyclase family members are randomly located on different chromosomes

Molecular cloning studies have elucidated the presence of multiple isoforms of mammalian adenylyl cyclase. So far, six different isoforms (I to VI) have been fully characterized. Comparison of their structural and biochemical characteristics suggests that the mammalian adenylyl cyclase family can be classified into four sub-families: type I, type III, type II/IV, and type V/VI. We have determined the chromosomal localization of these genes. Type I gene was assigned to chromosome 7, type III to chromosome 2, types II and IV to chromosomes 5 and 14, and types V and VI to chromosomes 3 and 12. Our results indicate that the different adenylyl cyclase isoforms, even within the same subfamily, are distributed randomly in the genome, in contrast to the chromosomal organization of other components within the same signaling pathway, such as catecholamine receptors and G proteins.     

Large-scale reconstruction of 3D structures of human chromosomes from chromosomal contact data

Chromosomes are not positioned randomly within a nucleus, but instead, they adopt preferred spatial conformations to facilitate necessary long-range gene–gene interactions and regulations. Thus, obtaining the 3D shape of chromosomes of a genome is critical for understanding how the genome folds, functions and how its genes interact and are regulated. Here, we describe a method to reconstruct preferred 3D structures of individual chromosomes of the human genome from chromosomal contact data generated by the Hi-C chromosome conformation capturing technique. A novel parameterized objective function was designed for modeling chromosome structures, which was optimized by a gradient descent method to generate chromosomal structural models that could satisfy as many intra-chromosomal contacts as possible. We applied the objective function and the corresponding optimization method to two Hi-C chromosomal data sets of both a healthy and a cancerous human B-cell to construct 3D models of individual chromosomes at resolutions of 1 MB and 200 KB, respectively. The parameters used with the method were calibrated according to an independent fluorescence in situ hybridization experimental data. The structural models generated by our method could satisfy a high percentage of contacts (pairs of loci in interaction) and non-contacts (pairs of loci not in interaction) and were compatible with the known two-compartment organization of human chromatin structures. Furthermore, structural models generated at different resolutions and from randomly permuted data sets were consistent.     

MicroRNA regulation constrains the organization of target genes on mammalian chromosomes

The regulation of microRNAs (miRNAs) is a complicated process requiring a large number of molecular events to be coordinated in both space and time. It is not known whether this complicated regulation process constrains the organization of target genes on mammalian chromosomes. We performed a genome-wide analysis to provide a better picture of chromosomal organization of miRNA target genes. Our results showed clustering of the target genes (TGs) of miRNAs on mammalian chromosomes, and further revealed that the particular gene organization is constrained by miRNA regulation. The clustering pattern of TGs provides an insight into the complexity of miRNA regulation.     

Mapping of the gene family for human heat-shock protein 90 alpha to chromosomes 1, 4, 11, and 14.

The HSP90 family of heat-shock proteins (encoded by genes for HSP90 alpha and beta) constitutes one of the major groups of proteins that are synthesized at increased rates in response to heat and other forms of stress. We previously isolated two distinct cDNA clones for HSP90 alpha from human peripheral blood lymphocytes and from HeLa cells transfected with the adenovirus E1A gene, respectively. To determine the organization of this complex multigene family in the human genome, we used three complementary approaches: Southern analysis of a panel of human/hamster somatic cell hybrids, molecular cloning of the cosmid HSP90 alpha clones from libraries prepared with DNAs from human lymphoblastoid cells, and in situ hybridization to human chromosomes. We demonstrate here that nucleotide sequences that encode HSP90 alpha map to human chromosomes 1q21.2-q22, 4q35, 11p14.1-p14.2, and 14q32.3. The chromosomal mapping of the loci, HSPCAL1, HSPCAL2, HSPCAL3, HSPCAL4, and the characterization of the respective genes should facilitate clarification of the organization of this gene family and lead to a better understanding of the biological functions of the gene product.     

The pig genome: compositional analysis and identification of the gene-richest regions in chromosomes and nuclei

The isochore organization of the mammalian genome comprises a general pattern and some special patterns, the former being characterized by a wider compositional distribution of the DNA fragments. The large majority of the mammalian genomes belong to the former, and only some groups, such as the Myomorpha sub-order of Rodentia, belong to the latter. Here we describe the compositional organization of the pig (Sus scrofa) genome that belongs to the general mammalian pattern. We investigated (i) the compositional distribution of the genes by analysis of their GC3 levels (the GC levels at the third codon positions), and (ii) the correlation between the GC3 value of orthologous genes from pig and other vertebrates (human, calf, mouse, chicken, and Xenopus). As expected, the highest gene concentration corresponded to the H3 isochore family, and the highest GC3 correlations were observed in the pig/human and pig/calf comparisons. Then we identified, by in situ hybridization of the GC-richest H3 isochores, the pig chromosomal regions endowed by the highest gene-density that largely corresponded to the telomeric chromosomal bands. Moreover, we observed that these gene-rich bands are syntenic with the previously identified GC-richest/gene richest H3+ bands of the human chromosomes. At the cell nucleus level, we observed that the gene-dense region corresponded to the more internal compartment, as previously found in human and avian cell nuclei.     

Genomic organization of evolutionarily correlated genes in bacteria: limits and strategies

The need for efficient molecular interplay in time and space within a cell imposes strong constraints that could be partially relaxed if relative gene positions along chromosomes were appropriate. Comparative genomics studies have demonstrated the short-scale conservation of gene proximity along bacterial chromosomes. Additionally, the long-range periodic positioning of evolutionarily correlated genes within Escherichia coli has recently been highlighted. To gain further insight into these different genetic organizations, we examined the compromise between chromosomal proximity and periodicity for all available eubacterial genomes by evaluating groups of evolutionarily correlated genes from a benchmark data set. In enterobacteria, strict chromosomal proximity is found to be limited to groups under 20 genes, whereas periodicity is significant in all groups over 50. The E. coli K12 genome bears 511 periodic genes (12% of the genome), whose orthologs are found to be periodic in all eubacterial phyla. These periodic genes predominantly function in macromolecular synthesis and spatial organization of cellular components. They are enriched in essential and housekeeping genes and tend to often be constitutively expressed. On this basis, it is argued that chromosomal proximity and periodicity are ubiquitous complementary genomic strategies that favor the build-up of local concentrations of co-functional molecules. In particular, the periodic layout may facilitate chromosome folding to spatially organize the construction of major cell components. The transition at 20 genes is reminiscent of the size of the longest operons and of topological microdomains. The range for which DNA neighborhood optimizes biochemical interactions might therefore be defined by DNA topology.     

Coordinate gene regulation during hematopoiesis is related to genomic organization

Gene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood. To examine whether gene regulation is related to a discernible pattern of genomic organization, we analyzed the linear arrangement of co-regulated genes along chromosomes and determined the organization of chromosomes during the differentiation of a hematopoietic progenitor to erythroid and neutrophil cell types. Our analysis reveals that there is a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. We suggest that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation.     

Functional bias and spatial organization of genes in mutational hot and cold regions in the human genome

The neutral mutation rate is known to vary widely along human chromosomes, leading to mutational hot and cold regions. We provide evidence that categories of functionally related genes reside preferentially in mutationally hot or cold regions, the size of which we have measured. Genes in hot regions are biased toward extracellular communication (surface receptors, cell adhesion, immune response, etc.), while those in cold regions are biased toward essential cellular processes (gene regulation, RNA processing, protein modification, etc.). From a selective perspective, this organization of genes could minimize the mutational load on genes that need to be conserved and allow fast evolution for genes that must frequently adapt. We also analyze the effect of gene duplication and chromosomal recombination, which contribute significantly to these biases for certain categories of hot genes. Overall, our results show that genes are located nonrandomly with respect to hot and cold regions, offering the possibility that selection acts at the level of gene location in the human genome.     

Novel gene acquisition on carnivore Y chromosomes

Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.     

Genome sizes and chromosomes in the basal metazoan Hydra

Hydras belong to one of the earliest eumetazoan animal groups, but to date very little is known about their genome sizes, gene numbers, and chromosomes. Here we provide genome size estimates and corresponding karyotypes for five Hydra species. Nuclear DNA contents were assessed by slide-based Feulgen microphotometry. Hydra oligactis possesses the largest genome of 1450 Mbp, followed by similar 1 C capacities in H. carnea (1350 Mbp), H. vulgaris (1250 Mpb) and H. circumcincta (1150 Mbp). The smallest genome of 380 Mbp was determined in H. viridissima. While the number of chromosomes is identical in all five Hydra species (2n = 30), the size of the chromosomes is strictly correlated to the size of the genome, with H. viridissima having conspicuously small chromosomes. The taxonomic and evolutionary significance of the C-value and chromosomal size variation in this ancient group of metazoans as well as its impact on genomic organization and forthcoming genome projects are discussed.     

Characterization of yeast artificial chromosomes containing interleukin genes on human chromosome 5.

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.     

Evolution of GABA(A) receptor diversity in the human genome

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.     

Physical mapping of the CC-chemokine gene cluster on the human 17q11. 2 region.

Chemokines are a family of small secreted proteins that are involved in the trafficking of leukocytes by acting on G-protein-coupled receptors. Specific chemokines are also implicated in the regulation of angiogenesis and mobilization of hematopoietic cell precursors. Chemokines are subdivided into four groups on the basis of the relative positions of their conserved cysteines. For the CC-chemokine group, in which the first two (of four) conserved cysteines are adjacent, 22 members have been described so far. In this work, we have analyzed the genomic organization of these genes. We first assigned the genes encoding CC-chemokines to chromosomal regions and organized their relative positioning by using two radiation hybrid panels. Fifteen CC-chemokine genes were shown to be clustered within the 17q11.2 region of the human genome. These genes appeared to be segregated into two subclusters separated by about 2. 25 Mb (9 cR). Contigs of bacterial artificial chromosomes (BAC) covering these two subclusters were subsequently isolated and the localizations of the CC-chemokine genes within these contigs determined. The relative positioning of the BAC clones was determined with the help of fluorescence hybridization on combed genomic DNA. The cluster organization of the various CC-chemokine genes in the genome was found to be grossly consistent with their structural similarities. This map of the CC-chemokine gene cluster should facilitate the determination of the full sequence of the chromosomal region.     

Double-minute chromosomes as megabase cloning vehicles.

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.     

Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization

One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.