Radiation Hybrid Mapping of the Two Highly Homologous Human–Variant pMCHL Genes by PCR–SSCP

When gene loci are very similar in sequence, as in gene families or multiple pseudogenes, it is difficult to determine the specific location of the individual genes. We show here that applying PCR–SSCP to a radiation hybrid panel allowed mapping and specific sequencing of two genes with only a few sequence differences. Human–variant forms of the promelanin-concentrating hormone (pMCH) gene are found in two locations in the genome, previously localized by FISH to 5p14 and 5q12–q13. Without prior knowledge of sequence variation between the loci, we observed a difference in migration pattern in PCR–SSCP, indicating the presence of at least one point of sequence divergence. PCR–SSCP of 93 samples from a human–hamster radiation hybrid panel revealed the location of the genes to be between markers WI-4804 and AFM225YC5 on chromosome 5p, and between markers WI-3133 and WI-4225 on chromosome 5q. Sequencing of the two 680-bp PCR products from the hybrid panel demonstrated 3 bases of sequence difference between the 5p and 5q locations.     

Mapping nonrecombining regions in barley using multicolor FISH

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.     

Dielectric Response of Aramid Fiber‐Reinforced PEEK

Dielectric spectroscopy was applied for the first time to aramid fiber‐reinforced PEEK, wherein the effect of the fiber on the dielectric response was examined for both amorphous and crystalline poly(ether ether ketone) (PEEK) over wide temperature and frequency ranges. Whereas the temperature behavior of the dielectric losses of the materials exhibited the typical α and β processes of PEEK, the specific effect of the fibers in the crystalline PEEK was revealed in shifting the α process to a higher temperature. The unique effect of the fibers was expressed by a significantly higher activation energy and lower dielectric strength for the α relaxation, reflecting a higher constraint level that is imposed by the fiber. It is proposed that this additional constraint is associated with fiber generated transcrystallinity.

Scanning electron micrograph of transverse fracture surface of crystallized unidirectional aramid fiber‐reinforced PEEK.     

High-Resolution Landmark Framework for the Sequence-Ready Mapping of Xq23–q26.1

We have established a landmark framework map over 20-25 Mb of the long arm of the human X chromosome using yeast artificial chromosome (YAC) clones. The map has approximately one landmark per 45 kb of DNA and stretches from DXS7531 in proximal Xq23 to DXS895 in proximal Xq26, connecting to published framework maps on its proximal and distal sides. There are three gaps in the framework map resulting from the failure to obtain clone coverage from the YAC resources available. Estimates of the maximum sizes of these gaps have been obtained. The four YAC contigs have been positioned and oriented using somatic-cell hybrids and fluorescence in situ hybridization, and the largest is estimated to cover approximately 15 Mb of DNA. The framework map is being used to assemble a sequence-ready map in large-insert bacterial clones, as part of an international effort to complete the sequence of the X chromosome. PAC and BAC contigs currently cover 18 Mb of the region, and from these, 12 Mb of finished sequence is available.     

Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis

We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosomal origin. The remaining four genes were mapped in a somatic cell hybrid panel. All chromosomal assignments except one were in agreement with human–horse ZOO-FISH data and revealed new and more detailed information on the equine comparative map. CLU was mapped by synteny to ECA2 while human–horse ZOO-FISH data predicted that CLU would be located on ECA9. The assignment of IL1RN permitted analysis of gene order conservation between HSA2 and ECA15, which identified that an event of inversion had occurred during the evolution of these two homologous chromosomes.     

Genome Mapping by Fluorescent Fingerprinting

The construction of sequence-ready maps of overlapping genomic clones is central to large-scale genome sequencing. We have implemented a method for fluorescent fingerprinting of bacterial clones to assemble contig maps. The method utilizes three spectrally distinct fluorescently tagged dideoxy ATPs to specifically label the HindIII termini in HindIII and Sau3AI restriction digests of clones that are multiplexed prior to electrophoresis and data collection. There is excellent reproducibility of raw data, improved resolution of large fragments, and concordance between the results obtained using this and the equivalent radioactive protocol. This method also allows detection of smaller overlaps between clones when compared to the analysis of restriction digests on nondenaturing agarose gels.     

Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes

In mice, the imprinted Igf2 gene (expressed from the paternal allele), which encodes a growth-promoting factor (IGF-II), is linked closely to the reciprocally imprinted H19 locus on chromosome 7. Also imprinted (expressed from the maternal allele) is the Igf2r gene on chromsome 17 encoding the type 2 IGF receptor that is involved in degradation of excess IGF-II. Double mutant embryos carrying a deletion around the H19 region and also a targeted Igf2r allele, both inherited maternally, have extremely high levels of IGF-II (7- and 11-fold higher than normal in tissues and serum, respectively) as a result of biallelic Igf2 expression (imprint relaxation by deletion of H19-associated sequence) in combination with lack of the IGF2R-mediated IGF-II turnover. This excess of IGF-II causes somatic overgrowth, visceromegaly, placentomegaly, omphalocele, and cardiac and adrenal defects, which are also features of the Beckwith–Wiedemann syndrome (BWS), a genetically complex human disorder associated with chromosomal abnormalities in the 11p15.5 region where the IGF2 gene resides. In addition, the double mutant mouse embryos exhibit skeletal defects and cleft palate, which are manifestations observed frequently in the Simpson–Golabi–Behmel syndrome, another overgrowth disorder overlapping phenotypically, but not genetically, with BWS.     

Mapping and organization of highly-repeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6×-triticale

Three families of highly repeated sequences from rye and the rRNA multigenes (NOR and 5S) have been mapped by FISH and C-banding, in chromosomes of triticale. The pSc119.2 probe showed interstitial hybridization in chromosome arms 1RS, 1RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL, and is very effective for chromosome identification of rye chromosomes in triticale. This sequence also hybridizes to the 4A, 5A and the seven B-genome wheat chromosomes. Simultaneous hybridization with the pSc119.2 and pTa794 (5S rRNA) is very useful to distinguish the metacentric chromosomes 2R and 3R. The pSc74 probe appears at interstitial sites in the long arm of the most heterobrachial chromosomes (5R and 6R). The three repetitive sequences of 120 bp, 480 bp, and 610 bp hybridize to telomeric regions in rye chromosomes. Different arrangements and complex organizations consisting of arrays of three or more family sequences were found. The results demonstrate a great variation in the relative arrangement of the repetitive sequences in the telomeres of the rye chromosomes. There were quantitative differences in each cytological marker between triticale lines in bothin situ labelling and C-banding, probably as the result of differences in the number and/or kind of repeat sequence.     

Lymphocyte transforamtion and leucocyte migration–inhibition by Australia antigen

The occurrence of a state of cell-mediated immune responsiveness to the Australia antigen following acute virus-B hepatitis has been demonstrated by the use of two in vitro methods—the lymphocyte transformation test and the leucocyte migration–inhibition test. Australia antigen-rich serum and purified Australia antigen were found to be effective agents in both tests. Close correlation was observed between the two tests. Persistent carriers of the Australia antigen give negative results with both tests.     

Familial complex 3q;10q rearrangement unraveled by subtelomeric FISH analysis

In recent years, subtelomeric rearrangements have been identified as a major cause of multiple congenital anomalies/mental retardation syndromes. Currently, more than 2,500 individuals with mental retardation have been tested and reported in whom subtelomeric rearrangements were detected ranging from 2% to 29%. Therefore, subtelomeric FISH analysis is indicated as a second tier test after high-resolution G-banding analysis in patients with otherwise unexplained developmental delay/mental retardation and/or multiple congenital anomalies. We describe a patient and her three maternal female cousins, all showing an undiagnosed MCA/MR syndrome, associated with the same complex subtelomeric rearrangement. Subtelomeric FISH testing performed between 3(1/2) and 18 years after the initial karyotype showed, in all four patients, distal trisomy 3q and distal monosomy 10q as follows: 46,XX,ish der(10)t(3;10)(q29;q26.3)mat(D10S2488+,D10S2490-, D3S1272+,D10Z1+). Parental subtelomeric FISH analysis showed that the proposita's mother and three of four brothers and one of two sisters had a cryptic balanced 3:10 telomere translocation. The three brothers with the balanced translocation were father to one each of the three proband's cousins. All four affected girls showed a similar phenotype with pre/postnatal growth retardation, microcephaly, severe developmental delay/mental retardation, poor/absent speech, and a distinct pattern of malformation. On examination there were coarsening of facial features with low fronto-temporal hairline; thick eyebrows; bilateral epicanthal folds; hypertelorism; prominent nose with squared nasal root and narrow alar base; low-set posteriorly rotated large ears with a prominent anthelix; high arched palate; prominent chin; hands/feet brachydactyly; bilateral squint; hypotonia; and muscle hypotrophy. A slow overall improvement was seen in all patients over time. To our knowledge, this complex subtelomeric rearrangement in our patients has never been reported so far. Monosomy 10q has recently been described either isolated or as part of a complex rearrangement involving telomeres other than the 3q. Trisomy 3q29 has not yet been reported, but our patients resembled cases with 3q26 trisomy suggesting that the critical region of duplication for this phenotype is in 3q29.     

Conserved requirement for EGF–CFC genes in vertebrate left–right axis formation

Specification of the left-right (L-R) axis in the vertebrate embryo requires transfer of positional information from the node to the periphery, resulting in asymmetric gene expression in the lateral plate mesoderm. We show that this activation of L-R lateral asymmetry requires the evolutionarily conserved activity of members of the EGF-CFC family of extracellular factors. Targeted disruption of murine Cryptic results in L-R laterality defects including randomization of abdominal situs, hyposplenia, and pulmonary right isomerism, as well as randomized embryo turning and cardiac looping. Similarly, zebrafish one-eyed pinhead (oep) mutants that have been rescued partially by mRNA injection display heterotaxia, including randomization of heart looping and pancreas location. In both Cryptic and oep mutant embryos, L-R asymmetric expression of Nodal/cyclops, Lefty2/antivin, and Pitx2 does not occur in the lateral plate mesoderm, while in Cryptic mutants Lefty1 expression is absent from the prospective floor plate. Notably, L-R asymmetric expression of Nodal at the lateral edges of the node is still observed in Cryptic mutants, indicating that L-R specification has occurred in the node but not the lateral plate. Combined with the previous finding that oep is required for nodal signaling in zebrafish, we propose that a signaling pathway mediated by Nodal and EGF-CFC activities is essential for transfer of L-R positional information from the node.