Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region

Summary The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplificiation of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in aMnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Physical mapping of a 950-kb region surrounding a locus (D10S102) tightly linked to the MEN2A gene.

We have constructed a long-range contig of cosmid and YAC clones around D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2A (MEN2A). With D10S102 as a starting point, a 360-kb cosmid contig was constructed by bidirectional genomic walking, and at least six fragments from these cosmids showed high sequence homology to other species. Five YAC clones were also isolated at the D10S102 locus, and they formed a contig covering 950 kb of genomic DNA. Furthermore, we obtained six RFLP systems from the contig, which will serve as new resources for fine-scale genetic linkage mapping of the MEN2A locus.     

Neural time-resolution depending on waveform of spikes.

A neural mechanism for detecting temporal coincidence in spike arrival is examined. The neurons fire when some spikes arrive simultaneously. The neurons of the electric fish can detect the coincidence in the microsecond range under hard temporal constraints: the width of spikes is more than 0.5 msec and the arrival time jitters on the scale of tens of microseconds. Since the synaptic connections between those neurons are electronic, the neural circuit is represented by a circuit composed of electric resistances. Computer simulation of behavior of the electric circuit model is presented to show that the nervous system can achieve the fine temporal sensitivity under the constraints. Analysis of the model shows that waveform of spikes is a critical condition to produce the sensitivity; peaks of spikes must be sharp. Also, the effect of the jitter of spike arrival is estimated to indicate that the coincidence detecting mechanism is tolerant of the jitter.     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.