Isolation of human and murine homologues of the Drosophila minibrain gene: human homologue maps to 21q22.2 in the Down syndrome "critical region".

The presence of an extra copy of human chromosome 21 (trisomy 21), especially region 21q22.2, causes many phenotypes in Down syndrome, including mental retardation. To study genes potentially responsible for some of these phenotypes, we cloned a human candidate gene (DYRK) from 21q22.2 and its murine counterpart (Dyrk) that are homologous to the Drosophila minibrain (mnb) gene required for neurogenesis and to the rat Dyrk gene (dual specificity tyrosine phosphorylation regulated kinase). The three mammalian genes are highly conserved, >99% identical at the protein level over their 763-amino-acid (aa) open reading frame; in addition, the mammalian genes are 83% identical over 414 aa to the smaller 542-aa mnb protein. The predicted human DYRK and murine Dyrk proteins both contain a nuclear targeting signal sequence, a protein kinase domain, a putative leucine zipper motif, and a highly conserved 13-consecutive-histidine repeat. Fluorescence in situ hybridization and regional mapping data localize DYRK between markers D21S336 and D21S337 in the 21q22.2 region. Northern blot analysis indicated that both human and murine genes encode approximately 6-kb transcripts. PCR screening of cDNA libraries derived from various human and murine tissues indicated that DYRK and Dyrk are expressed both during development and in the adult. In situ hybridization of Dyrk to mouse embryos (13, 15, and 17 days postcoitus) indicates a differential spatial and temporal pattern of expression, with the most abundant signal localized in brain gray matter, spinal cord, and retina. The observed expression pattern is coincident with many of the clinical findings in trisomy 21. Its chromosomal locus (21q22. 2), its homology to the mnb gene, and the in situ hybridization expression patterns of the murine Dyrk combined with the fact that transgenic mice for a YAC to which DYRK maps are mentally deficient suggest that DYRK may be involved in the abnormal neurogenesis found in Down syndrome.

Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.

We present a fluorescent-PCR-based technique to assay genomic sequence copy number and transcriptional abundance. This technique relies on the ability to follow fluorescent PCR progressively in real time during the exponential phase of the reaction so that quantitative PCR is accomplished. We demonstrated the ability of this technique to quantitate both known deletions and amplifications of loci that have been measured previously by other methods, and to measure transcriptional abundance. Using an efficient variant of the fluorescent-PCR technology, we can monitor transcription semiquantitatively. The ability to detect all amplifications and deletions at any single copy locus by PCR makes this the technique of choice to assay genomic sequence copy number anomalies in birth defects and cancers. The ability to detect variations in transcript abundance enables this technique to fashion a time and tissue analysis of transcription.

Identification and analysis of the human and murine putative chromatin structure regulator SUPT6H and Supt6h.

We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively. The human and murine SPT6 homologues are virtually identical, as they share >98% identity and >99% similarity at the protein level. The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively. There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper. These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development. Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7. 0-kb transcript that was expressed constitutively. The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization. These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure.

Expressed sequence tags from the long arm of human chromosome 21.

We isolated expressed sequence tags (ESTs) on the long arm of chromosome 21. The ESTs were mapped by PCR using a monochromosomal somatic-cell mapping panel. Of a total of 55 cDNAs, 30 mapped back uniquely to chromosome 21, 7 mapped back to other chromosomes including chromosome 21, 8 mapped back to chromosomes other than 21, and 10 could not be assigned using this methodology. The 30 chromosome 21-specific markers so isolated represent useful EST markers. A rapid PCR-based method was used to delineate the expression pattern of these 30 pairs in different tissues.

Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis.

We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was > or = 650 base pairs and the average initial complexity of the given libraries was 10(6). We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development.

Transcription patterns of sequences on human chromosome 21.

The polymerase chain reaction (PCR) was used to screen embryonic, fetal and adult human cDNA libraries for transcription on chromosome 21q22.1-->q22.3. Seventy-three pairs of oligonucleotide primers on chromosome 21, used previously to screen a fetal brain cDNA library, were applied to analyze 41 different cDNA libraries. Only phage eluate (and therefore no DNA isolation) was required for this sensitive screening. Sixty primer pairs were positive with at least one cDNA library, indicating that the majority of primers were derived from transcribed sequences. Even with our most complex human fetal brain cDNA library, we detected only 57% (34/60) of transcribed sequences, illustrating the need to screen multiple human cDNA libraries to determine if transcription occurred. Since only 3/73 clones were present in only one cDNA library, the vast majority of transcribed sequences are present in more than one tissue.

Physical location of the human immunoglobulin lambda-like genes, 14.1, 16.1, and 16.2.

The human immunoglobulin lambda-like (IGLL) genes, which are homologous to the human immunoglobulin lambda (IGL) light chain genes, are expressed only in pre-B cells and are involved in B cell development. Three IGLL genes, 14.1, 16.1, and 16.2 are present in humans as opposed to one, lambda 5 (Igll), found in the mouse. To precisely map the location of the human IGLL genes in relation to each other and to the human IGL gene locus, at 22q11.1-2, a somatic cell hybrid panel and pulsed field gel electrophoresis (PFGE) were used. Hybridization with a lambda-like gene-specific DNA probe to somatic cell hybrids revealed that these genes reside on 22q11.2 between the breakpoint cluster region (BCR) and the Ewing sarcoma breakpoint at 22q12 and that gene 16.1 was located distal to genes 14.1 and 16.2. Gene 14.1 was found by PFGE to be proximal to 16.2 by at least 30 kilobases (kb). A 210 kb Not I fragment containing genes 14.1 and 16.2 is adjacent to a 400 kb Not I fragment containing the BCR locus, which is just distal to the IGL-C (IGL constant region) genes. We have determined that the IGLL genes 14.1 and 16.2 are approximately 670 kb and 690 to 830 kb distal, respectively, to the 3'-most IGL-C gene in the IGL gene locus, IGL-C7. We thus show the first physical linkage of the IGL and the IGLL genes, 14.1 and 16.2. We discuss the relevance of methylation patterns and CpG islands to expression, and the evolutionary significance of the IGLL gene duplications. Consistent with the GenBank nomenclature, these human IGLL genes will be referred to as IGLL1 (14.1), IGLL2 (16.2), and IGLL3 (16.1), reflecting their position on chromosome 22, as established by this report.

Analysis of proteins expressed at the time of murine organogenesis.

Two-dimensional electrophoretograms were prepared from wild-type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed in mouse embryos development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament-associated protein and tropomyosin-4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy).

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.

Analysis of proteins synthesized by 9.5 day mouse embryos: determination of cardiac and noncardiac proteins.

To catalog polypeptides that were specific to developing hearts, we separated 35S-methionine-labeled 9.5 day mouse embryos into cardiac and noncardiac (carcass) components. Two-dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or increased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac-specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac-specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac-specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two-dimensional gels.

Developmental appearance of proteins identified by two-dimensional gel electrophoresis in mouse gonadal tissue.

Gonadal protein patterns of the mouse were studied during fetal development by two-dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post-coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.

DNA sequences surrounding the centromere of chromosome 21.

The mapping and sequencing of two clones that surround the centromere of chromosome 21 are presented. These clones specify the most proximal known low-order repeat on 21p (p21-7D) and the most proximal known single-copy sequence on 21q (pUT-B37 at locus D21S120).

Characterization of an unusual and complex chromosome 21 rearrangement using somatic cell genetics and cloned DNA probes.

In a previous case of a newborn infant with typical Down syndrome, chromosome analysis indicated the presence of an unusual and complex translocation of chromosome 21. The patient's cells contained one normal chromosome 21 and a rearranged, F group-sized submetacentric chromosome. This abnormal chromosome appeared to involve duplication of the distal portion of 21q with translocation to the short arm, and a deletion of C-band-positive centromeric heterochromatin. Using linearly ordered cloned DNA probes, we report the detailed molecular examination of this abnormal chromosome, which has been isolated on a hamster background in a hybrid cell line. Both short arm and pericentromeric sequences are present on this chromosome, as well as distal 21q sequences. However, a substantial portion of proximal 21q is deleted. The distal boundary of this deleted section can be pinpointed within the region between two loci (D21S8 and D21S54), a distance of about 5,000 kb. This study illustrates the power of using precisely mapped, linearly ordered DNA probes to characterize this type of rearrangement. In addition, this hybrid cell line can also be used as a member of a mapping panel to map DNA sequences regionally on chromosome 21.

Molecular mechanism in the formation of a human ring chromosome 21.

We have characterized the structural rearrangements of a chromosome 21 that led to the de novo formation of a human ring chromosome 21 [r(21)]. Molecular cloning and chromosomal localization of the DNA regions flanking the ring junction provide evidence for a long arm to long arm fusion in formation of the r(21). In addition, the centromere and proximal long arm region of a maternal chromosome 21 are duplicated in the r(21). Therefore, the mechanism in formation of the r(21) was complex involving two sequential chromosomal rearrangements. (i) Duplication of the centromere and long arm of one maternal chromosome 21 occurred forming a rearranged intermediate. (ii) Chromosomal breaks in both the proximal and telomeric long arm regions on opposite arms of this rearranged chromosome occurred with subsequent reunion producing the r(21).

Confirmation of assignment of the human alpha 1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3.

The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, alpha-, beta-, and gamma-crystallins, and each class represents a multigene family. The alpha-crystallin gene family consists of alpha 1-crystallin (CRYA1) and alpha 2-crystallin (CRYA2) genes (previously designated alpha A- and alpha B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human alpha 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human alpha 2-crystallin gene and does not cross-hybridize to alpha 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the alpha 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.

Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus.

The amyloid beta protein has been identified as an important component of both cerebrovascular amyloid and amyloid plaques of Alzheimer's disease and Down syndrome. A complementary DNA for the beta protein suggests that it derives from a larger protein expressed in a variety of tissues. Overexpression of the gene in brain tissue from fetuses with Down syndrome (trisomy 21) can be explained by dosage since the locus encoding the beta protein maps to chromosome 21. Regional localization of this gene by both physical and genetic mapping places it in the vicinity of the genetic defect causing the inherited form of Alzheimer's disease.

A somatic cell hybrid with a single human chromosome 22 corrects the defect in the CHO mutant (Ade-I) lacking adenylosuccinase activity.

Adenine-requiring Chinese hamster ovary (CHO-K1) auxotrophs of the complementation group Ade-I were hybridized with various human cells, and hybrids were isolated under selective conditions in which retention of the complementing gene on the human chromosome is necessary for survival. Ade-I cells are deficient in adenylosuccinase activity. This enzyme carries out two independent, but similar, steps of purine biosynthesis: the removal of a fumarate from succinylaminoimidazole carboxamide ribotide to produce aminoimidazole carboxamide ribotide and the removal of fumarate from adenylosuccinate to produce AMP. These are the 9th and 13th steps of adenylate biosynthesis, respectively. Analysis of hybrids by cytogenetics and by Southern blot techniques using chromosome 22-specific DNA probes, one of which encodes an antigen expressed in human fetal brain, indicated that human chromosome 22 was 100% concordant for growth without adenine. One hybrid subclone, isolated after two successive rounds of subcloning, was found to be capable of growth without adenine; the only human chromosome present was 22. In addition, segregants that had lost the ability to grow in adenine-free media had also lost human chromosome 22. These results suggest that the human gene for adenylosuccinase resides on chromosome 22.

Assignment of a third purine biosynthetic gene (glycinamide ribonucleotide transformylase) to human chromosome 21.

Using a series of human-hamster hybrid cell lines, a gene coding for glycinamide ribonucleotide transformylase was mapped to human chromosome 21. The availability of hybrids containing only portions of chromosome 21 allowed the gene to be assigned to the region between the q11.2 and the q22.2 bands, inclusive. Differentiation of human and hamster glycinamide ribonucleotide transformylase was accomplished via an immunoprecipitation assay that employed a polyclonal antibody raised against the human enzyme.