We have cloned the cDNA encoding human liver glycogen phosphorylase (glycogenosis type VI) from a fetal brain cDNA library. Liver (L) and muscle (M) phosphorylase cDNA probes were used to determine ...the relative abundance of mRNA encoding the L- and M-isozymes of phosphorylase in human fetal and adult tissues. The transcript encoding the M-isozyme is 3.4 kb; the L-isozyme transcript is 3.3 kb. Transcriptional expression of the L-isozyme in human and primate tissues was found to differ from the isozyme's reported tissue specificity in non-primate mammals. Furthermore, using degenerate oligonucleotide probes to two different coding regions of M-phosphorylase, a novel 4.1-kb transcript was demonstrated to be present in human fetal and adult brain.
The central feature of Alzheimer's disease is a remarkably specific loss of cognitive functions in affected individuals. The evidence that links Alzheimer's disease neurodegeneration and pathology ...with APP and/or its Aβ-containing derivatives is the early finding that the APP gene is on chromosome 21, for virtually all individuals trisomic for this chromosome will show AD-like neurodegeneration by the age of 40. It is found that PC12 cells transfected with a retroviral recombinant expressing the carboxyterminal 100 amino acids of APP die when they are induced to acquire a neuronal phenotype with nerve growth factor (NGF), while transfectants expressing exogenous APP-695 or Aβ differentiate normally without cell loss when exposed to NGF. Transplantation of APP-C100-transfected PC12 cells into the brains of newborn mice results in neuropathology. Transgenic mice expressing APP-C100 in the brain display Alzheimer's disease-like neuropathology. Expression of APP-C100 in the brain leads to massive neurodegeneration in aged transgenic mice.
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.
The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein
(beta APP) of which three alternatively spliced versions of 695, 751, ...and 770 amino acids have been described. A fourth beta
APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa
cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57,
115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion
of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following
this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's
disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established
cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar
to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108
kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment
of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass.
The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and
the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A previously uncharacterized 22-kDa Ca2+-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, ...calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca2+, calexcitin bound GTP and possessed GTPase activity. Calexcitin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca2+and the absence of endogenous Ca2+/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 ± 6 pS K+channels. Calexcitin thus appears to directly regulate K+channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca2+-activated signaling molecule that increases cellular excitability, which would in turn increase Ca2+influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca2+.
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Recombinant DNA techniques provide new approaches to the diagnosis and analysis of inherited human diseases associated with mental retardation. Examples of such diseases include the Lesch-Nyhan ...syndrome, phenylketonuria, the Fragile X syndrome, Down syndrome, and those associated with deletions or duplications of subchromosomal regions, e.g., the proximal short arm of human chromosome #15. For a limited but increasing number of diseases, the DNA sequences responsible for the phenotype (e.g., sequences coding for abnormal proteins) can be isolated directly. In many other cases, DNA segments mapping near genes responsible for diseases of interest can be isolated, e.g., from recombinant phage libraries enriched for specific regions of the genome by metaphase chromosome flow-sorting and then used in molecular linkage studies to "track" the abnormal gene in a pedigree. Both the necessary technology and the methods for its application continue to improve, and the impact of recombinant DNA studies in the field of mental retardation should increase markedly in the very near future.
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