Heterochromatin,gene position effect and gene silencing

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.     

Chicken deoxyribonuclease: purification,characterization, gene cloning and gene expression

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn²⁺ and Mg²⁺ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu²⁺, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca²⁺ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.     

The evolution of the plastid chromosome in land plants: gene content, gene order, gene function

This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.     

The relationship between gene dosage,gene expression,and sex in Drosophila

In Drosophila, the ratio of the number of X chromosomes to sets of other chromosomes initiates a series of events which result in sexual differentiation. In addition, this ratio establishes dosage compensation, a mechanism which equalizes the products of X-linked genes in males and females. The present review discusses possible genetic entities responsible for the interpretation of chromosomal sex and subsequent sex-mediated regulation during development.     

Owl monkey gene mapping: the assignment of gene loci for catalase, beta-globin gene cluster, HRAS1, insulin, and parathyroid hormone

Using somatic cell genetics and Southern blot hybridization, we have mapped five structural genes in the owl monkey, coding for catalase (CAT), the beta-globin gene cluster (HBBC), c-Ha-ras 1 (HRAS1), insulin (INS), and parathyroid hormone (PTH). All five loci are mapped to chromosome 19 of karyotype VI (2n = 49,50) of the owl monkey; CAT, HBBC, INS, and PTH can be assigned to chromosome 4 of karyotype V (2n = 46), while CAT and HBBC can be assigned to chromosome 2 of karyotype III (2n = 53). Using in situ hybridization, the CAT gene was precisely mapped on the mid-region and the beta-globin gene cluster on the telomeric end of chromosome 2q(K-III). Our results provide significant insight into the evolutionary history of these gene loci. While these loci are separated into at least two major segments in rodents such as the mouse, our results suggest conservation of a single chromosome arm among higher primates.     

The natural history of class I primate alcohol dehydrogenases includes gene duplication, gene loss, and gene conversion

Background

Gene duplication is a source of molecular innovation throughout evolution. However, even with massive amounts of genome sequence data, correlating gene duplication with speciation and other events in natural history can be difficult. This is especially true in its most interesting cases, where rapid and multiple duplications are likely to reflect adaptation to rapidly changing environments and life styles. This may be so for Class I of alcohol dehydrogenases (ADH1s), where multiple duplications occurred in primate lineages in Old and New World monkeys (OWMs and NWMs) and hominoids.

Methodology/Principal Findings

To build a preferred model for the natural history of ADH1s, we determined the sequences of nine new ADH1 genes, finding for the first time multiple paralogs in various prosimians (lemurs, strepsirhines). Database mining then identified novel ADH1 paralogs in both macaque (an OWM) and marmoset (a NWM). These were used with the previously identified human paralogs to resolve controversies relating to dates of duplication and gene conversion in the ADH1 family. Central to these controversies are differences in the topologies of trees generated from exonic (coding) sequences and intronic sequences.

Conclusions/Significance

We provide evidence that gene conversions are the primary source of difference, using molecular clock dating of duplications and analyses of microinsertions and deletions (micro-indels). The tree topology inferred from intron sequences appear to more correctly represent the natural history of ADH1s, with the ADH1 paralogs in platyrrhines (NWMs) and catarrhines (OWMs and hominoids) having arisen by duplications shortly predating the divergence of OWMs and NWMs. We also conclude that paralogs in lemurs arose independently. Finally, we identify errors in database interpretation as the source of controversies concerning gene conversion. These analyses provide a model for the natural history of ADH1s that posits four ADH1 paralogs in the ancestor of Catarrhine and Platyrrhine primates, followed by the loss of an ADH1 paralog in the human lineage.     

Evolution of gene families based on gene duplication, loss, accumulated change, and innovation.

We introduce and analyze a simple discrete probabilistic model of genome evolution. It is based on four fundamental evolutionary events: gene duplication, loss, accumulated change and innovation. We call it the DLCI model. It is the first such model rigorously analyzed. The focus of the paper is on the size distribution of gene families. We derive formulae for the equilibrium of gene family sizes, and show that they follow a logarithmic distribution. We also consider a disjoint union of DLCI models and present empirical results for bacterial genomes.     

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus.

Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.     

A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.     

Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene

Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5′ and 3′ remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.     

Thecab-m7 gene: a light-inducible,mesophyll-specific gene of maize

Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclearcab multigene family encoding the chlorophylla- andb-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3 probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designatedcab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. Thiscab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts ofcab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of thecab-m7 gene resemble those found in the 5-flanking regions of some other plant genes.