DNA deletions in mild and severe Becker muscular dystrophy

Summary The DNA of 33 patients diagnosed as suffering from Becker muscular dystrophy (BMD) has been probed with cloned DNA sequences from Xp21, known to reveal DNA deletions in patients suffering from the more severe Duchenne muscular dystrophy (DMD). Two BMD cases showed clear deletions. A third case gave aberrant band sizes, which further analysis showed to be caused by a small deletion. This suggests that deletions in DXS164 occur approximately as frequently in BMD as they do in DMD. Of the two cases showing large deletions, one is at the severe end of the Becker clinical spectrum, whilst the other is a classical Becker-type dystrophy. The fact that loci defined by probes commonly deleted in classical DMD patients are also deleted in BMD patients of varying severity is strong additional evidence that these disorders are allelic, and further justifies the use of probes with defined linkage relationships to DMD also being used for counselling in BMD families.     

The molecular genetics of haemophilia A: screening for point mutations in the factor VIII gene using the restriction enzyme TaqI

Summary A combination of Southern blotting and the analysis of polymerase chain reaction (PCR) amplified DNA fragments was used to screen the factor VIII genes of 527 haemophilia A patients for point mutations within TaqI restriction sites. Since this directed search strategy yielded only four gene lesions, it was concluded that its efficacy is less than that originally predicted. One novel point mutation was however found in a moderately severe haemophiliac; a CGA (Arg) to CTA (Leu) transversion at codon 2209, an evolutionarily conserved residue in the C2 domain of the factor VIII protein. The remaining three detected lesions, CGA (Arg)TGA (Term) transitions at codons 2116, 2147 and 2307, respectively, have been reported before and are consistent with recurrent mutation at these hypermutable sites. A number of TaqI restriction site polymorphisms/rare variants were also noted. These variants appear to be population-specific but are nevertheless potentially useful in individual cases as intragenic markers for carrier detection and antenatal diagnosis.     

DNA inversions in phages and bacteria

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.     

Efficient algorithms for analyzing segmental duplications with deletions and inversions in genomes

Background  

Segmental duplications, or low-copy repeats, are common in mammalian genomes. In the human genome, most segmental duplications are mosaics comprised of multiple duplicated fragments. This complex genomic organization complicates analysis of the evolutionary history of these sequences. One model proposed to explain this mosaic patterns is a model of repeated aggregation and subsequent duplication of genomic sequences.     

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions in recombinant plasmids in Escherichia coli

The ability to clone a variety of sequences with varying capabilities of adopting non-B structures (left-handed Z-DNA, cruciforms or triplexes) into three loci of pBR322 was investigated. In general, the inserts were stable (non-deleted) in the EcoRI site (an untranslated region) of pBR322. However, sequences most likely to adopt left-handed Z-DNA or triplexes in vivo suffered deletions when cloned into the BamHI site, which is located in the tetracycline resistance structural gene (tet). Conversely, when the promoter for the tet gene was altered by filling-in the unique HindIII or ClaI sites, the inserts in the BamHI site were not deleted. Concomitantly, the negative linking differences of the plasmids were reduced. Also, inserts with a high potential to adopt Z-DNA conformations were substantially deleted in the PvuII site of pBR322 (near the replication origin and the copy number control region), but were less deleted if the tet promoter was insertion-mutated. The deletion phenomena are due to the capacity of these sequences to adopt left-handed Z-DNA or triplexes in vivo since shorter inserts, less prone to form non-B DNA structures, or random sequences, did not exhibit this behavior. Sequences with the potential to adopt cruciforms were stable in all sites under all conditions. These results reveal a complex interrelationship between insert deletions (apparently the result of genetic recombination), negative supercoiling, and the formation of non-B DNA structures in living Escherichia coli cells.     

A local algorithm for DNA sequence alignment with inversions

A dynamic programming algorithm to find all optimal alignments of DNA subsequences is described. The alignments use not only substitutions, insertions and deletions of nucleotides but also inversions (reversed complements) of substrings of the sequences. The inversion alignments themselves contain substitutions, insertions and deletions of nucleotides. We study the problem of alignment with non-intersecting inversions. To provide a computationally efficient algorithm we restrict candidate inversions to theK highest scoring inversions. An algorithm to find theJ best non-intersecting alignments with inversions is also described. The new algorithm is applied to the regions of mitochondrial DNA ofDrosophila yakuba and mouse coding for URF6 and cytochrome b and the inversion of the URF6 gene is found. The open problem of intersecting inversions is discussed.     

The myotonic dystrophy type 1 triplet repeat sequence induces gross deletions and inversions

The capacity of (CTG.CAG)n and (GAA.TTC)n repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreich's ataxia, respectively. Long (CTG.CAG)n (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6-1.8 kbp were found as well as inversions. Shorter repeat tracts (where n = 0 or 17) were essentially inert, as observed for the (GAA.TTC)176-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30-50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1-8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.     

A severe form of human combined immunodeficiency due to mutations in DNA ligase IV

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and alphabeta T cells 10-fold, whereas gammadelta T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of gammadelta T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype.     

Patterns of bleeding in adolescents with severe haemophilia A.

Eighty-two boys with severe haemophilia A who spent some time at Lord Mayor Treloar College during 1973-7 were studied. All episodes of bleeding that occurred during term time were recorded, along with the number of transfusions. The bleeding frequency among these boys, most of them aged 10-17 years, increased steadily from 8,31 episodes/100 days in 1973 to 12,63 episodes/100 days in 1977. At the same time there was a steady fall in bleeding frequency with age. Altogether 24% of bleeding episodes were into the elbow joint, 22% into the knee, and 15% into the ankle. As the boys grew older the proportion of bleeding episodes in the legs declined and that in the arms increased. The overall results reflect the fact that special schools now see only the severest cases of haemophilia. The pattern of bleeding during adolescence suggests that concepts of management of arm bleeding need modifying.     

Transposon 10 promoted deletions and inversions in the transfer genes of R100-1

Summary The replication of the ColE1 plasmid was studied in extracts from E. coli dnaG mutants. It was found that the synthesis of the complementary strands of ColE1 DNA can be carried out in these extracts in two consecutive steps: (1) synthesis of the leading L strand independent of the dnaG function, and (2) synthesis of the lagging H strand depending upon addition of wild-type dnaG protein. In contrast to L strand synthesis, the latter reaction is insensitive to rifampicin and novobiocin. Both synthetic pathways are however blocked by antiserum directed against dnaB protein. This indicates an additional role of the dnaB protein in duplex DNA replication besides assisting the dnaG protein in the priming of lagging strand synthesis. The T7 gene-4 protein acting in conjunction with T7 DNA polymerase can substitute for both the function of the dnaB and dnaG protein. It is concluded that plasmid replication proceeds by a semi-discontinuous mechanism.     

Human DNA polymerase beta mutations allowing efficient abasic site bypass

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.