Academic literature on the topic 'Eukaryotic gene; Genes; Chromosomal domain'

While colocalization within a bacterial operon enables coexpression of the constituent genes, the mechanistic logic of clustering of nonhomologous monocistronic genes in eukaryotes is not immediately obvious. Biosynthetic gene clusters that encode pathways for specialized metabolites are an exception to the classical eukaryote rule of random gene location and provide paradigmatic exemplars with which to understand eukaryotic cluster dynamics and regulation. Here, using 3C, Hi-C, and Capture Hi-C (CHi-C) organ-specific chromosome conformation capture techniques along with high-resolution microscopy, we investigate how chromosome topology relates to transcriptional activity of clustered biosynthetic pathway genes inArabidopsis thaliana. Our analyses reveal that biosynthetic gene clusters are embedded in local hot spots of 3D contacts that segregate cluster regions from the surrounding chromosome environment. The spatial conformation of these cluster-associated domains differs between transcriptionally active and silenced clusters. We further show that silenced clusters associate with heterochromatic chromosomal domains toward the periphery of the nucleus, while transcriptionally active clusters relocate away from the nuclear periphery. Examination of chromosome structure at unrelated clusters in maize, rice, and tomato indicates that integration of clustered pathway genes into distinct topological domains is a common feature in plant genomes. Our results shed light on the potential mechanisms that constrain coexpression within clusters of nonhomologous eukaryotic genes and suggest that gene clustering in the one-dimensional chromosome is accompanied by compartmentalization of the 3D chromosome.

Typically, eukaryotic nuclei contain 10–30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain “choice” appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3–4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments (∼5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic “neighborhoods.”

Parental imprinting is a gamete-specific modification that distinguishes the paternal and maternal chromosomes in higher eukaryotes, resulting in allele-specific changes in chromatin organization, transcription and replication. One example of parental imprinting in humans is revealed by two distinct genetic diseases, Prader-Willi syndrome (PWS) and Angelman syndrome (AS) which both map to chromosome 15q11-13. PWS is caused by the absence of a paternal contribution to 15q11-13, while AS results from the lack of a maternal copy of the region. Within this chromosomal subregion lies a cluster of GABAA receptor β3 and α5 subunit genes (GABRB3 and GABRA5) which are separated by about 100 kb and arranged in opposite transcritional orientations (Figure 1). Allele-specific asynchronous DNA replication has previously been found to be associated with imprinted chromosomal regions.In order to further study the association between DNA replication and imprinting, allele-specific replication was assayed by fluorescence in situ hybridization (FISH). Biotin-labeled phage probes detected by FITC hybridized to each chromosome as either a singlet (unreplicated state) or a doublet (replicated state). Cells demonstrating asynchronous replication (one singlet and one doublet) for each probe are shown in Figure 2.

The study spatial chromosome structure and chromosome folding in the interphase cell nucleus is an important challenge of world science. Detection of eukaryotic genome regions that physically interact with each other could be done by modern sequencing technologies. A basic method of chromosome folding by total sequencing of contacting DNA fragments is HI-C. Long-range chromosomal interactions play an important role in gene transcription and regulation. The study of chromosome interactions, 3D (three-dimensional) genome structure and its effect on gene transcription allows revealing fundamental biological processes from a viewpoint of structural regulation and are important for cancer research. The technique of chromatin immunoprecipitation and subsequent sequencing (ChIP-seq) make possible to determine binding sites of transcription factors that regulate expression of eukaryotic genes; genome transcription factors binding maps have been. The ChIA-PET technology allows exploring not only target protein binding sites, but also pairs of such sites on proximally located and interacting with each other chromosomes co-located in three-dimensional space of the cell nucleus. Here we discuss the principles of the construction of genomic maps and matrices of chromosome contacts according to ChIA-PET and Hi-C data that capture the chromosome conformation and overview existing software for 3D genome analysis including in house programs of gene location analysis in topological domains.

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe. By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.

The Drosophila chorion genes encode the major protein components of the chorion (eggshell) and are arranged in two clusters in the genome. To meet the demand for rapid chorion synthesis, Drosophila ovary follicle cells amplify the chorion gene clusters approximately 80-fold. Amplification proceeds through repeated firing of one or more DNA replication origins located near the center of each gene cluster. Hypomorphic mutant alleles of the chiffon gene cause thin, fragile chorions and female sterility, and were found to eliminate chorion gene amplification. Null alleles of chiffon had the additional phenotypes of rough eyes and thin thoracic bristles: phenotypes often associated with disruption of normal cell cycle. The chiffon locus was cloned by chromosomal walking from the nearby cactus locus. A 6.5 kb transcript was identified and confirmed to be chiffon by sequencing of mutant alleles and by phenotypic rescue with genomic transformation constructs. The protein predicted by translation of the 5.1 kb chiffon ORF contains two domains related to the S. cerevisiae Dbf4 regulator of DNA replication origin firing and cell cycle progression: a 44 residue domain designated CDDN1 (43% identical) and a 41 residue domain designated CDDN2 (12% identical). The CDDN domains were also found in the S. pombe homolog of Dbf4, Dfp1, as well as in the proteins predicted by translation of the Aspergillus nimO gene and specific human and mouse clones. The data suggest a family of eukaryotic proteins related to Dbf4 and involved in initiation of DNA replication.

Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.

Chromosomes of higher eukaryotes are thought to be organized into a series of discrete and topologically independent higher-order domains. In addition to providing a mechanism for chromatin compaction, these higher-order domains are thought to define independent units of gene activity. Implicit in most models for the folding of the chromatin fiber are special nucleoprotein structures, the domain boundaries, which serve to delimit each higher-order chromosomal domain. We have used an "enhancer-blocking assay" to test putative domain boundaries for boundary function in vivo. This assay is based on the notion that in delimiting independent units of gene activity, domain boundaries should be able to restrict the scope of activity of enhancer elements to genes which reside within the same domain. In this case, interposing a boundary between an enhancer and a promoter should block the action of the enhancer. In the experiments reported here, we have used the yolk protein-1 enhancer element and an hsp70 promoter:lacZ fusion gene to test putative boundary DNA segments for enhancer-blocking activity. We have found that several scs-like elements are capable of blocking the action of the yp-1 enhancer when placed between it and the hsp70 promoter. In contrast, a MAR/SAR DNA segment and another spacer DNA segment had no apparent effect on enhancer activity.

The protein phosphatase (PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs (i.e., segmental and dispersed) of Gossypium hirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.

Multicellular eukaryotes have evolved a range of mechanisms for immune recognition. A widespread family involved in innate immunity are the NACHT-domain and leucine-rich-repeat-containing (NLR) proteins. Mammals have small numbers of NLR proteins, whereas in some species, mostly those without adaptive immune systems, NLRs have expanded into very large families. We describe a family of nearly 400 NLR proteins encoded in the zebrafish genome. The proteins share a defining overall structure, which arose in fishes after a fusion of the core NLR domains with a B30.2 domain, but can be subdivided into four groups based on their NACHT domains. Gene conversion acting differentially on the NACHT and B30.2 domains has shaped the family and created the groups. Evidence of positive selection in the B30.2 domain indicates that this domain rather than the leucine-rich repeats acts as the pathogen recognition module. In an unusual chromosomal organization, the majority of the genes are located on one chromosome arm, interspersed with other large multigene families, including a new family encoding zinc-finger proteins. The NLR-B30.2 proteins represent a new family with diversity in the specific recognition module that is present in fishes in spite of the parallel existence of an adaptive immune system.

Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.