Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Functional cloning of drug resistance genes from retroviral cDNA libraries

To improve the curative success of chemotherapy, it will be essential to understand the molecular basis of drug resistance (DR) and sensitivity. We have developed a cell culture system that enables the functional cloning of mammalian DR genes based on phenotypic selection after overexpression of mammalian retroviral cDNA libraries and validated our system using the anticancer drug cisplatin. ERCC1-deficient and therefore cisplatin-hypersensitive mouse embryonic fibroblast target cells were transduced with a human placenta retroviral cDNA library. Subsequent cisplatin selection yielded 20 DR clones, each containing a recurring human ERCC1 gene. Surprisingly, nine of these clones contained 5'-truncated ERCC1 sequences that required alternative splicing of the vector sequence to encode a functional ERCC1 protein. The usage of cryptic splice sites in the vector sequence should be taken into consideration when interpreting results from retroviral gene expression applications, and might have consequences for the safe application of retroviral constructs in gene therapy.     

Relationship of micro-and minisatellites in the human genome

Micro-and minisatellites constitute an essential part of DNA with low sequence complexity and perform a number of important functions. The TandemSWAN program was used to search the human genome for tandem repeats with a length of a repeated unit to 70 bp, including repeats with a large number of nucleotide substitutions. It was shown that, for a significant fraction of the program-found minisatellites with a repeat unit length less than 25 bp, a shorter repeated motif can be discerned in this sequence, which is often similar to the sequence of microsatellites occurring widely in the human genome. A model of hierarchical origin of minisatellites in the human genome was proposed.     

Two highly polymorphic minisatellites from the pseudoautosomal region of the human sex chromosomes.

Two pseudoautosomal loci DXYS15 and DXYS17 from the pairing region of the human sex chromosomes display a high variability with at least eight alleles each. The structural elements responsible for the polymorphisms have been isolated and sequenced. In both cases the variations result from DNA rearrangements occurring in tandemly repeated sequences (minisatellites) of 21-29 nucleotides for DXYS15 and 28-33 nucleotides for DXYS17. At reduced stringency, the DXYS15 minisatellite detects other hypervariable sequences located in other parts of the genome and hence represents a new family of minisatellites. In contrast to most other known hypervariable families, the DXYS15 hypervariable sequence displays a very high AT content.     

Relationship between micro- and minisatellites in the human genome

Micro- and minisatellites constitute an essential part of DNA with a low sequence complexity and carry several important functions. A search for tandem repeats in the human genome with a length of a repeat unit of up to 70 bp, including repeats with a great number of nucleotide substitutions, has been performed using the TaadeaSWAN program. It was shown that, for a considerable number of minisatellites with the length of the repeating unit of less than 25 nt, a shorter repeating motif can be distinguished in the sequence of this repeat, which often is similar to the sequence of minisatellites widely occurring in the human genome. A model of hierarchic origination of minisatellites in the human genome is suggested.     

Clustering of hypervariable minisatellites in the proterminal regions of human autosomes

Six of the human minisatellites detected by DNA fingerprint probes have been localized by in situ hybridization to human metaphase chromosomes. These hypervariable loci are not dispersed at random in the human genome, but show preferential, though not exclusive, localization to terminal G-bands of human autosomes. Two of the proterminal minisatellites are very closely linked to other variable loci. Sequence analysis of one of these additional minisatellites suggests that the two linked minisatellites arose by independent amplification of different repeat units. The proterminal regions of human autosomes may therefore be rich in minisatellites, analogous to the pseudoautosomal terminal pairing region of human sex chromosomes that is similarly abundant in hypervariable minisatellites.     

Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.     

Comparison of minisatellites.

In the class of repeated sequences that occur in DNA, minisatellites have been found polymorphic and became useful tools in genetic mapping and forensic studies. They consist of a heterogeneous tandem array of a short repeat unit. The slightly different units along the array are called variants. Minisatellites evolve mainly through tandem duplications and tandem deletions of variants. Jeffreys et al. (1997) devised a method to obtain the sequence of variants along the array in a digital code and called such sequences maps. Minisatellite maps give access to the detail of mutation processes at work on such loci. In this paper, we design an algorithm to compare two maps under an evolutionary model that includes deletion, insertion, mutation, tandem duplication, and tandem deletion of a variant. Our method computes an optimal alignment in reasonable time; and the alignment score, i.e., the weighted sum of its elementary operations, is a distance metric between maps. The main difficulty is that the optimal sequence of operations depends on the order in which they are applied to the map. Taking the maps of the minisatellite MSY1 of 609 men, we computed all pairwise distances and reconstructed an evolutionary tree of these individuals. MSY1 (DYF155S1) is a hypervariable locus on the Y chromosome. In our tree, the populations of some haplogroups are monophyletic, showing that one can decipher a microevolutionary signal using minisatellite maps comparison.     

Flow cytometry-assisted cloning of specific sequence motifs from complex 16S rRNA gene libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.     

RAPD-based screening of genomic libraries for positional cloning.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.     

Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

Background  

Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated.     

Selection of tumor-specific internalizing human antibodies from phage libraries

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.     

Selection of functional human antibodies from retroviral display libraries

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10³-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.     

Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.     

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.     

Construction of cloned libraries from RNA of human fetal tissues

We have constructed libraries of recombinant DNA plasmids containing sequences complementary to polyadenylated RNA from a variety of human midtrimester fetal tissues. The bacterial colonies containing these plasmids have been grown and replicated on nitrocellulose filters in a manner that facilitates permanent storage, rapid screening, and transportability to other laboratories. We screened a portion of the library for the presence of repetitive sequences and found that approximately 20% of the clones contain repetitive sequences. We have also shown that some clones contain nonrepetitive sequences. Pools of recombinant cDNA-containing plasmids devoid of repetitive sequences have been constructed to permit the chromosomal localization of a variety of actively transcribed sequences. The construction of such large, tissue-specific clone banks should facilitate the direct isolation, mapping, and characterization of normal and abnormal human genes.     

Construction of fifteen human chromosome-specific DNA libraries from flow-purified chromosomes

We report the construction of 15 human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow sorting and the DNA was extracted and cleaved with HindIII before cloning into the lambda vector Charon 21A. A sensitive miniblot hybridization method was used to monitor the physical and biochemical steps in the cloning procedure. Using this method, we have developed a highly efficient protocol for producing large numbers of recombinant phage from 0.2-1.0 X 10(6) sorted chromosomes. DNA from the following chromosomes was cloned: #4, 6, 8, 9, 11, 13, 14 + 15, 16, 17, 18, 19, 20, 21, 22 and Y. These libraries are available to the scientific research community and will be valuable in the genetic analysis of the human genome.