Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Telomeric repeat sequences

Chromosomes not only carry transcribed genes and their regulatory DNA sequences, but also contain regions that are required for the stability and maintenace of the chromosome as a unit. These include centromeres, telomeres and origins of replication. It is clear for replication origins and centromeres that the positions of these chromosomal organelles are determined by sites of the appropriate DNA sequences, but also that functional performance requires one or more contributing proteins. Telomeres are also structurally complex, with one or more DNA components, including simple telomeric repeats and more complex telomere-associated sequences, as well as one or more specific proteins that recognize these sequences. Accumulating evidence suggests that the simple telomeric repeats are required in most, but not all species, although they are not sufficient to determine the chromosomal position of a telomere.     

Identification of repeat structure in large genomes using repeat probability clouds

The identification of repeat structure in eukaryotic genomes can be time-consuming and difficult because of the large amount of information (3 × 10⁹ bp) that needs to be processed and compared. We introduce a new approach based on exact word counts to evaluate, de novo, the repeat structure present within large eukaryotic genomes. This approach avoids sequence alignment and similarity search, two of the most time-consuming components of traditional methods for repeat identification. Algorithms were implemented to efficiently calculate exact counts for any length oligonucleotide in large genomes. Based on these oligonucleotide counts, oligonucleotide excess probability clouds, or “P-clouds,” were constructed. P-clouds are composed of clusters of related oligonucleotides that occur, as a group, more often than expected by chance. After construction, P-clouds were mapped back onto the genome, and regions of high P-cloud density were identified as repetitive regions based on a sliding window approach. This efficient method is capable of analyzing the repeat content of the entire human genome on a single desktop computer in less than half a day, at least 10-fold faster than current approaches. The predicted repetitive regions strongly overlap with known repeat elements as well as other repetitive regions such as gene families, pseudogenes, and segmental duplicons. This method should be extremely useful as a tool for use in de novo identification of repeat structure in large newly sequenced genomes.     

TROLL--tandem repeat occurrence locator

SUMMARY: Tandem Repeat Occurrence Locator (TROLL), is a light-weight Simple Sequence Repeat (SSR) finder based on a slight modification of the Aho-Corasick algorithm. It is fast and only requires a standard Personal Computer (PC) to operate. We report running times of 127 s to find all SSRs of length 20 bp or more on the complete Arabdopsis genome--approx. 130 Mbases divided in five chromosomes--using a PC Athlon 650 MHz with 256 MB of RAM. AVAILABILITY: TROLL is an open source project and is available at http://finder.sourceforge.net.     

Unstable nucleotide repeat minireview series: a molecular biography of unstable repeat disorders

Expansion of an unstable nucleotide repeat is a mutational mechanism that is apparently unique to humans and is known to cause a variety of neurological disorders. This collection of minireviews examines several of these unstable repeats, focusing on those where there is considerable molecular information on how the mutation alters function.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers.

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.     

Evolution of the spectrin repeat

We now know that the evolution of multidomain proteins has frequently involved genetic duplication events. These, however, are sometimes difficult to trace because of low sequence similarity between duplicated segments. Spectrin, the major component of the membrane skeleton that provides elasticity to the cell, contains tandemly repeated sequences of 106 amino acid residues. The same repeats are also present in α-actinin, dystrophin and utrophin. Sequence alignments and phylogenetic trees of these domains allow us to interpret the evolutionary relationship between these proteins, concluding that spectrin evolved from α-actinin by an elongation process that included two duplications of a block of seven repeats. This analysis shows how a modular protein unit can be used in the evolution of large cytoskeletal structures.     

Fostering repeat donations in Ghana

IntroductionMost African countries are challenged in recruiting and retaining voluntary blood donors by cost and other complexities and in establishing and implementing national blood policies. The availability of replacement donors who are a cheaper source of blood has not enhanced repeat voluntary donor initiatives.MethodsAn overview of activities for recruiting and retaining voluntary blood donors was carried out. Donor records from mobile sessions were reviewed from 2002 to 2008.Results and discussionA total of 71,701 blood donations; 45,515 (63.5%) being voluntary donations with 11,680 (25%) repeat donations were collected during the study period. Donations from schools and colleges contributed a steady 60% of total voluntary whilst radio station blood drives increased contribution from 10 to 27%. Though Muslim population is less than 20%, blood collection was above the 30-donation cost-effectiveness threshold with a repeat donation trend reaching 60%. In contrast Christian worshippers provided <25 unit/session and 30% repeat donations. Repeat donation trends amongst school donors and radio blood drives were 20% and 70% respectively.ConclusionRepeat donations rates have been variable amongst different blood donor groups in Kumasi, Ghana. The impact of community leaders in propagating altruism cannot be overemphasized. Programs aiming at motivating replacement donors to be repeat donors should be developed and assessed.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.     

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.