David M. Kurnit

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.

Human chromosome 21-encoded cDNA clones

We have employed two strategies to isolate random cDNA clones encoded by chromosome 21. In the first approach, a cDNA library representing expressed genes of WA17, a mouse-human somatic cell hybrid carrying chromosome 21 as its sole human chromosome, was screened with total human DNA to identify human chromosome 21-specific cDNAs. The second approach utilized previously characterized single-copy genomic fragments from chromosome 21 as probes to retrieve homologous coding sequences from a human fetal brain cDNA library. Six cDNA clones on chromosome 21 were obtained in this manner. Two were localized to the proximal long arm of chromosome 21, two to the distal portion of the long arm, and one to the region of 21q22 implicated in the pathology of Down syndrome.

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).

The 724 family of DNA sequences is interspersed about the pericentromeric regions of human acrocentric chromosomes

We describe the organization of the complex, interspersed 724 family of DNA sequences that is distributed in multiple copies about the pericentromeric region of human acrocentric chromosomes. 724 family members were isolated using an efficient recombination-based assay for nucleotide sequence homology to screen a human genomic library. Eight related but distinct 724 family members were isolated that hybridized to a total of 20 different human-genomic EcoRI DNA fragments spanning 100,000 base pairs. In contrast with tandemly clustered satellite and ribosomal DNA sequences also located on the short arms of human acrocentric chromosomes, 724 family members are interspersed. No evidence for local interspersion or homology between 724 family members and ribosomal or satellite DNA sequences was found. Juxtaposition of the complex 724 family to the nucleolus organizer region was a recent event in primate evolution. The unique organization of 724 family members on each of the five human acrocentric chromosomes indicates that the 724 family continues to evolve within the human karyotype.

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.

Genetics of Congenital Heart Malformations: A Stochastic Model†

A stochastic model is proposed to explain how alterations in the properties of developing endocardial cells could control the outgrowth of endocardial cushions in normal persons, in subjects from families with a predisposition to congenital heart defects, and in subjects with trisomy 21. Normal and abnormal outgrowth of the endocardial cushions of the atrioventricular (AV) canal were modeled by computer simulations. Computer simulations depicted not only the sequence of normal AV valve development, but also illustrated how increased cellular adhesiveness of fibroblasts from the endocardial cushions of the AV canal--which we have observed in vitro among cultured cells from Down syndrome abortuses--may result in AV canal defects. The stochastic model so elaborated demonstrates how single gene changes may result in abnormalities in only a proportion of subjects carrying mutant alleles, yielding inheritance patterns characterized previously as being multifactorial in origin.

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.

Molecular dysmorphology: an approach to Down's syndrome.

I wish to propose the following hypothesis to explain how gene dosage for genes encoded by chromosome 21 could yield birth defects in Down’s syndrome: InGreased dosage for genes on chromosome 21 increases the adhesiveness among trisomy 21 cells during embryogenesis, resulting in specific deficiencies of outgrowth of embryonic tissues that are later recognizable as characteristic congenital anomalies. The fundamental question posed by Down’s syndrome is how one can go from the karyotypic abnormality of trisomy 21 to the phenotypic abnormalities of Down’s syndrome. Down’s syndrome differs from more easily understood Mendelian syndromes: In Down’s syndrome the alteration of genetic material is quantitative, not qualitative. Thus the genetic material is normal in Down’s syndrome, and the error is “too much of a good thing.” Several years ago, I began to look at the cascade of abnormalities that one might expect from trisomy 21. I affirmed that dosage for genes on chromosome 21 occurred at the transcriptional level in fibroblasts from subjects with monosomy 21, normal subjects (disomy 21), and those with Down’s syndrome (trisomy 21). Yoram Groner and colleagues have also affirmed the transcriptional basis for gene dosage using a cloned DNA probe from a gene, superoxide dismutase, encoded by chromosome 21. Several researchers, including Charles Epstein, David Cox, David Patterson, and W. Ted Brown, have shown that the transcriptional increase is reflected at the translational level, so that increased dosage for proteins encoded by genes on chromosome 21 is observed. To describe the concept of gene dosage and trisomy 21, I use the analogy of three factories turning out three fleets of cars, instead of the normal two factories turning out two fleets of cars. The next, more difficult question is: How does having too much of this good thing result in developmental anomalies? As one looks at the list of anomalies associated with Down’s syndrome, three points stand out: specificity, variability, and hypoplasia. The specificity is great enough so that individuals with Down’s syndrome may be diagnosed by inspection, both externally (by the characteristic facies) and internally (by the characteristic endocardia1 cushion defects, the shape of the iliac bones, and propensity to specific defects such as duodenal obstruction). To illustrate the variability, only 40% of children with Down’s syndrome have clinically significant congenital heart