Linking of the human immunoglobulin VK and JKCK regions by chromosomal walking.

The linking of the human VK and JKCK gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between JKCK and the telomer show that none of the major VK gene clusters is located downstream of CK. The distance between the VK and JK genes was found to be 23 kb. The JK proximal VK gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to JKCK and can therefore rearrange only by an inversion mechanism. This finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step.     

Reciprocal recombination products of VK-JK joining reactions in human lymphoid cell lines.

The recombination process that joins a VK to a JK segment of an immunoglobulin gene generates a second, reciprocal recombination product called f fragment. In this second product the regions flanking the VK and JK segments in the germline are joined in a head to head fashion. We now analysed f fragments in the human lymphoid cell lines Daudi, JI and IARC/BL41. All three f fragments contain JK1 flanks; the VK derived moiety of f Daudi and f41 could be traced back to known germline VK genes. There is a precise head to head joining of the heptanucleotide signal sequences in f Daudi and fJI while in f41 six nucleotides are present between the signal sequences. In contrast to the VK-JK recombination products, the f fragments were found to lack somatic mutations. The structures of the f fragments are discussed in the context of the VK-JK rearrangement mechanism.     

Physical and genetic map of the chromosome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.

A combined physical and genetic map of the cyanobacterium Synechocystis sp. strain PCC 6803 chromosome was constructed. An estimated genome size of 3.82 Mb was obtained by summing the sizes of 25 MluI or 40 NotI fragments seen by pulsed-field electrophoresis. The order of the restriction fragments was determined by using two independent experimental approaches: pulsed-field fragment hybridization and linking clone analysis. The relative positions of 30 known genes or gene clusters were localized.     

Structural features of transposed human VK genes and implications for the mechanism of their transpositions.

The genes encoding the variable, joining and constant regions of human immunoglobulin light chains have been localized to the short arm of chromosome 2. However, several VK genes lie outside of the locus: a single copy cluster of five VK genes is located on chromosome 22; an isolated but amplified VkI gene is found on chromosome 1; and several isolated VkI genes are on as-yet-unidentified chromosomes other than chromosome 2. Vk genes not contained within the kappa locus are termed orphons. We have attempted to gain insight into the mechanism of transposition of both the chromosome 22 cluster and the several amplified VkI genes by searching in the kappa locus for a parent copy of the former, and by analyzing the junctions between transposed VKI-containing segments and adjacent non-amplified regions. The chromosome 22 orphon cluster must have been non-duplicatively transposed. Sequence features at the junctions of this and other orphon regions are direct and inverted repeats, and, in one case, an Alu repeat. These unusual features may have predisposed the orphon regions to transposition by serving as target sites for enzymes involved in recombination.     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

Immunoglobulin genes of different subgroups are interdigitated within the VK locus

The variable regions of immunoglobulins are encoded by multigene families which are rearranged during B-cell differentiation. These families were classified in groups and subgroups based on their amino acid sequences. Genes belonging to a distinct subgroup are believed to occur in the genome within clusters. We are investigating the organization of human variable region genes of the kappa type (VK genes, ref. 1) in the germline and found now for the first time that VK sequences of three of the four different subgroups are interdigitated within the VK locus. We present evidence for the interspersion of two VKIII genes and a VKII pseudogene within an array of five VKI genes. All eight VK sequences are arranged in the same orientation. An evolutionary model for the generation of this 'mixed cluster' is discussed.     

Evidence for non-homologous end joining and non-allelic homologous recombination in atypical NF1 microdeletions

NF1 microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by non-allelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while the remaining deletions show unusual breakpoints. We performed high-resolution FISH analysis of 18 NF1 microdeleted patients with the aims of mapping non-recurrent deletion breakpoints and verifying the presence of additional recombination-prone architectural motifs. This approach allowed us to obtain the sequence of the first junction fragment of an atypical deletion. By conventional FISH, we identified 16 patients with REP-mediated common deletions, and two patients carrying atypical deletions of 1.3 Mb and 3 Mb. Following fibre-FISH, we identified breakpoint regions of 100 kb, which led to the generation of several locus-specific probes restricting the atypical deletion endpoint intervals to a few kilobases. Sequence analysis provided evidence of small blocks of REPs, clustered around the 1.3-Mb deletion breakpoints, probably involved in intrachromatid non-allelic homologous recombination (NAHR), while isolation and sequencing of the 3-Mb deletion junction fragment indicated that a non-homologous end joining (NHEJ) mechanism is implicated.M. Venturin and C. Gervasini contributed equally to the study     

A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes

By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3' from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.     

Genome-wide signatures of 'rearrangement hotspots' within segmental duplications in humans

The primary objective of this study was to create a genome-wide high resolution map (i.e., >100 bp) of 'rearrangement hotspots' which can facilitate the identification of regions capable of mediating de novo deletions or duplications in humans. A hierarchical method was employed to fragment segmental duplications (SDs) into multiple smaller SD units. Combining an end space free pairwise alignment algorithm with a 'seed and extend' approach, we have exhaustively searched 409 million alignments to detect complex structural rearrangements within the reference-guided assembly of the NA18507 human genome (18× coverage), including the previously identified novel 4.8 Mb sequence from de novo assembly within this genome. We have identified 1,963 rearrangement hotspots within SDs which encompass 166 genes and display an enrichment of duplicated gene nucleotide variants (DNVs). These regions are correlated with increased non-allelic homologous recombination (NAHR) event frequency which presumably represents the origin of copy number variations (CNVs) and pathogenic duplications/deletions. Analysis revealed that 20% of the detected hotspots are clustered within the proximal and distal SD breakpoints flanked by the pathogenic deletions/duplications that have been mapped for 24 NAHR-mediated genomic disorders. FISH Validation of selected complex regions revealed 94% concordance with in silico localization of the highly homologous derivatives. Other results from this study indicate that intra-chromosomal recombination is enhanced in genic compared with agenic duplicated regions, and that gene desert regions comprising SDs may represent reservoirs for creation of novel genes. The generation of genome-wide signatures of 'rearrangement hotspots', which likely serve as templates for NAHR, may provide a powerful approach towards understanding the underlying mutational mechanism(s) for development of constitutional and acquired diseases.     

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.     

The pseudoautosomal regions, SHOX and disease

The pseudoautosomal regions represent blocks of sequence identity between the mammalian sex chromosomes. In humans, they reside at the ends of the X and Y chromosomes and encompass roughly 2.7 Mb (PAR1) and 0.33 Mb (PAR2). As a major asset of recently available sequence data, our view of their structural characteristics could be refined considerably. While PAR2 resembles the overall sequence composition of the X chromosome and exhibits only slightly elevated recombination rates, PAR1 is characterized by a significantly higher GC content and a completely different repeat structure. In addition, it exhibits one of the highest recombination frequencies throughout the entire human genome and, probably as a consequence of its structural features, displays a significantly faster rate of evolution. It therefore represents an exceptional model to explore the correlation between meiotic recombination and evolutionary forces such as gene mutation and conversion. At least twenty-nine genes lie within the human pseudoautosomal regions, and these genes exhibit 'autosomal' rather than sex-specific inheritance. All genes within PAR1 escape X inactivation and are therefore candidates for the etiology of haploinsufficiency disorders including Turner syndrome (45,X). However, the only known disease gene within the pseudoautosomal regions is the SHORT STATURE HOMEBOX (SHOX) gene, functional loss of which is causally related to various short stature conditions and disturbed bone development. Recent analyses have furthermore revealed that the phosphorylation-sensitive function of SHOX is directly involved in chondrocyte differentiation and maturation.