Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans. CRKL maps within the common deletion region for DGS/VCFS (ref. 2) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products. Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol-/- embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.

Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5' -* 3' toward the chromosomal end. The terminal repeats in yeast are the only elements necessary for telomere function in this organism. To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line. In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria. Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional. Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.

All telomeres which have been studied consist of an array of simple G/C rich repeats. Human telomeres were shown to share sequence similarity with those of lower eukaryotes by cross-hybridization and human telomeric sequences have been cloned by complementation of telomere function in yeast. Analysis of human telomeric sequences cloned in this way is described here. The terminal part of the cloned human telomeric DNA consists of an array of simple repeats, principally of the sequence TTAGGG and derivatives. The very terminal part consists of yeast-type telomeric repeats which suggests that the human telomeric sequences have acted as a primer for the addition of additional telomeric repeats in the yeast. Subterminal sequences are shared between a number of clones and in situ data shows that these subterminal sequences are present at several different chromosomal ends. Related sequences are present at internal as well as telomeric positions. Differences in the hybridization patterns of subterminal sequences in somatic compared to germ-line tissues are described which indicate differential modification of these sequences during development.

Drosophila ovo͞svb (dovo) is required for epidermal cuticle͞den-ticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of dovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Here, we provide biochemical evidence that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those that dOvo binds to, further supporting the notion that mOvo1a and dOvo are genetically and biochemically homologous proteins. Additionally, we show that the movo1 promoter is activated by the lymphoid enhancer factor 1 (LEF1)͞␤-catenin complex, a transducer of wnt signaling. Collectively, our findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg͞wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies.hair follicle ͉ wnt ͉ wg ͉ mouse ovo1

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We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

Key terms: Background correction, CCD camera., filters, fluorochromes, image quality assessment. intensity normalization, pixel shift correction, relative copy number karyotypeComparative Genomic Hybridization (CGH) analysis (4) has rapidly established itself as a powerful method for analyzing whole genomic DNA (3, 5 , 6, 10, 14, 15), and many researchers are interested in acquiring their own CGH analysis capability. Although some chromosomal imbalances, e.g. high copy number amplifications, can be identified by conventional microscopy, comprehensive interpretation of CGH requires digital image analysis. However, at the time of writing there are no comrnercially available software packages which come close to the capabilities of the in-house systems used by the various research teams which have been at the forefront of CGH developments and application (9, 11, 13).At a recent workshop o n the topic of "CGH Imaging" (organised by the EC Concerted Action on Automation of Molecular Cytogenetic Analyses, and held at the MRC Human Genetics Unit, Edinburgh, 2-3 June 1994), three discussion sessions were devoted to specifying what could be considered to be an "adequate provision" of hardware and analysis software, and what guidelines should be used in the interpretation of CGH data. These are respectively the topics of the three sections of this report, which summarises the consensus that was reached at that workshop.We hope that these recommendations will prove to be useful for prospective users of CGH. This technical note is not intended to be an introduction to CGH, nor a sur. vey of the literature on the subject, nor is it concerned with preparative techniques (which are discussed in de.. tail in Refs. 7, 8). Readers are referred to original source articles for detailed information on biological preparation protocols, digital microscopy, and analysis software; and also for further discussion of the issues concerning interpretation of the results of CGH analyses, and limitations of the technique. HARDWARE REQUIREMENTS Microscope OpticsRecent microscope models from the major manufacturers, equipped for epi-fluorescence, are adequate. The objective should be "plan" and apochromatic with a high numerical aperture. Some comniercially available apochromatic objectives are not suitable for CGH analysis since they do not transmit UV excitation. Some recent "fluar" objectivcs have developed high levels of autofluorescence after short periods of use; users should check for lens autofluorescence regularly and replace the objective when necessary. Mercury arc lamp light sources are adequate if they are stable, and can be aligned to give uniform illumination without chromatic variation. Recent microscope models hare improved collector lenses that correct for chromatic aberrations of the illumination, but problems may be experienced with older models. FiltersFilter sets should be selected to minimize the crosstalk betwcen fluorochromes. Ideally, this would be achieved by using fluorochromes with completely separated excitation a...

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)‐conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cy tometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to ch romosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above‐average kinetochore fluorescence did not produce decisive radiation dosimetry results.