Genomic Scaffolds Research Articles

Expressed sequence tags: alternative or complement to whole genome sequences?

Over three million sequences from approximately 200 plant species have been deposited in the publicly available plant expressed sequence tag (EST) sequence databases. Many of the ESTs have been sequenced as an alternative to complete genome sequencing or as a substrate for cDNA array-based expression analyses. This creates a formidable resource from both biodiversity and gene-discovery standpoints. Bioinformatics-based sequence analysis tools have extended the scope of EST analysis into the fields of proteomics, marker development and genome annotation. Although EST collections are certainly no substitute for a whole genome scaffold, this ‘poor man's genome’ resource forms the core foundations for various genome-scale experiments within the as yet unsequenceable plant genomes.

Cutting edge: Monarch-1: a pyrin/nucleotide-binding domain/leucine-rich repeat protein that controls classical and nonclassical MHC class I genes.

Proteins containing a limited number of N-terminal motifs followed by nucleotide-binding domain and leucine-rich repeat regions are emerging as important regulators for immunity. A search of human genome scaffold databases has identified a large family of known and unknown genes, which we have recently called the CATERPILLER (caspase recruitment domain, transcription enhancer, r(purine)-binding, pyrin, lots of leucine repeats) gene family. This work describes the characterization of a new member, Monarch-1. Monarch-1 has four different splice forms due to the differential splicing of leucine-rich repeat motifs. It is expressed in cells of myeloid-monocytic origin. Affymetrix microarrays and small interfering RNA were used to elucidate the downstream effects of Monarch-1 expression in cells including those of myeloid-monocytic origin. These analyses show that Monarch-1 enhances nonclassical and classical MHC class I expression at the level of the promoter, RNA, and protein expression.

Efficient Recovery of Centric Heterochromatin P-Element Insertions in Drosophila melanogaster

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication.

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.

Complex Study of Drosophila Mutants in the agnostic Locus: A Model for Coupling Chromosomal Architecture and Cognitive Functions

After the devastation of genetics in our country, Academician Leon A. Orbeli has provided an opportunity for the studies on evolutionary conservatism of genes controlling the main properties of the higher nervous activity and conditioning. For the last few years, determination and bioinformatic analysis of genome sequences in the plant, worm, Drosophila, and human genome have revealed, indeed, a high interspecies homology of genes. Studies on Drosophila mutants have shown that components of intracellular signalization systems regulating neuronal functions and gene expression are organized in supramolecular complexes. It has become evident that the chromosomal architecture predetermines the appearance of deletions, duplications, insertions, and translocations and, therefore, plays an important role not only in evolution but also in generating human pathological syndromes with multiple manifestations, including cognitive dysfunctions. There appeared a new approach, comparative genomics, that allows revealing functions of human disease genes on the basis of their sequence homology to the known Drosophila gene with various well-studied mutant phenotypes. For this reason, the Drosophila genes should be saturated with mutant phenotypes, and these are to be studied in comparison with the chromosomal architecture. Our complex behavioral and molecular-genetic study of spontaneous, induced, and P-insertional mutations in the Drosophila agnostic locus and the bioinformatic analyses of genomic sequences has allowed us to assign the locus to the Drosophila genomic scaffold AE003489 from the 11AB X-chromosomal region that contains the CG1848 gene coding for LIM-kinase 1. Mutations, insertions, and deletions in the agnostic locus lead to an increased activity of Ca2+/calmodulin-dependent PDE1, resistance to ether, an inactivator of synaptic transmission, impairments of the brain structures, learning and memory defects in conditioned courtship suppression paradigm, alterations in sound production and in structural-functional chromosomal organization. Therefore, the agnostic locus represents a model to study the human Williams syndrome with multiple dysfunctions due to a contiguous deletion in the 7q11.23 spanning 17 genes, among them the gene for LIM-kinase 1 presumed to be responsible for cognitive defects. The Williams syndrome is considered to be a most efficient model to study human cognition, human genome organization, and evolution.

A look at optical mapping

Whole-genome shotgun optical mapping is one of the new genome-scale technologies that is dramatically changing our approaches towards understanding bacterial genome structure. Optical mapping quickly provides a picture of the number of chromosomes and extrachromosomal elements in a bacterium, without the uncertainties of data obtained by the traditional pulsed-field gel electrophoresis (PFGE). For genome sequencers, optical mapping is quickly becoming an invaluable tool because it provides a high-resolution genome scaffold that facilitates verification of sequence assemblies1.