Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0–4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294–304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

A highly divergedβ1 exon in theDR region of the human MHC: Sequence and evolutionary implications

TheDR subregion of the human major histocompatibility complex from aDR4 haplotype includes the well-characterizedDR _ga ,DR4 DR(MT3) andDR genes. In addition, the region between theDR and the proximalDR(MT3) genes contains several copies of conserved DR -related sequences. These repeated elements, numbered II, III, and IV, include the DR signal sequence and a region located further upstream. Further examination of these conserved sequences showed that DR first intron sequences are present at the 3 ends of these repeats. Progressively longer portions of the DR first intron are conserved from repeat II to repeat IV, producing a gradient of conservation. The most complete repeat element of repeats II, III, and IV is associated with a lone1 exon (DR ₁). Upon sequencing, (DR ₁). was found to contain several deleterious mutations, indicating that it is nonfunctional. (DR ₁). has accumulated a large number of replacement substitutions and mutations at positions which are invariant in1 domains from expressedDR genes: 77.8% of the nucleotide substitutions were replacement substitutions, and 41.5 % of the amino acids at invariant positions have been altered. Calculations based on these figures suggest thatDR 1 may have become inactive approximately 25 million years ago. There are, however, two histidine residues within a variable region which are unique toDR 1 and theDR4 gene, suggesting that they represent a gene pair which probably evolved by duplication of a singleDR chain gene.     

The distribution and interaction of haemoglobin variants and the β thalassaemia gene in Liberia

In a population survey in Liberia, West Africa, 12 major tribes were examined for the prevalence of Hb S, Hb C, and the beta thalassaemia (beta Thal) gene. Hb C is rare; Hb S and beta Thal occur in polymorphic frequencies. The distribution of both genes shows an inverse correlation. The beta Thal trait was diagnosed by quantitation of Hb A2 on DE 52-microchromatography. This method proved to be reliable and useful for mass screening.     

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.     

NMR structure of the 5′ splice site in the group IIB intron Sc.ai5γ—conformational requirements for exon–intron recognition

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5′ exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5′ exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5′ exon and the intron. Here, we present the NMR solution structures of the d3′ hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg²⁺ ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.     

The chromosomal distribution of repetitive DNA sequences within the human β globin gene cluster

Summary The chromosomal distribution and degree of repetitiveness of the sequences within the human globin gene cluster has been studied by in situ hybridisation. A genomic recombinant, H\G1, which contains 15.9 thousand base pairs (kb) of DNA inserted into charon 4A, was used as a template for the [³H] complementary RNA used for hybridisation. The inserted DNA contains sequences 4.7 kb to the 5 side of the gene and continues through the and genes to a site 2.8 kb to the 3 side of the gene. Two highly repetitive sequences, which are distributed evenly over all the chromosomes, have been identified within this DNA. Another somewhat less repetitive sequence has been identified between the and genes.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

Lateral gene transfer in eukaryotes: tip of the iceberg or of the ice cube?

Lateral gene transfer (LGT) is the transmission of genes, sometimes across species barriers, outwith the classic vertical inheritance from parent to offspring. LGT is recognized as an important phenomenon that has shaped the genomes and biology of prokaryotes. Whether LGT in eukaryotes is important and widespread remains controversial. A study in BMC Biology concludes that LGT in eukaryotes is neither continuous nor prevalent and suggests a rule of thumb for judging when apparent LGT may reflect contamination.See research article: http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-0315-9.     

Population study of a sequence polymorphism in intron 2 of the human β-globin gene

The distribution of a nucleotide polymorphism in intron 2 of the -globin gene (IVS-2 nt 666 C > T was examined in populations in southern Germany and Cameroon. The allelic frequencies were 0.86 for T and 0.14 for C in southern Germany and 0.87 for T and 0.13 for C in Cameroon, respectively.     

The 5′-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms

Introns are important for regulating gene expression. BmAPN4, which has a 5′-UTR upstream intron (5UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5UI was cloned and named as P3P+5UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5UI. Transgenic P3+5UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5UI silkworms was 64% higher than in P3 silkworms, indicating the 5UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5UI affected the level and site of expression. The 5UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5UI and transgenic P2+5UI silkworms. Expression patterns were the same for P2P+5UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.