Unique Distribution of <Emphasis Type="Italic">GAL</Emphasis> Genes on Chromosome XI in the Yeast <Emphasis Type="Italic">Saccharomyces naganishii</Emphasis>

A region of DNA extending from GAL7 to GAL1 was cloned in the yeast Saccharomyces naganishii. Sequence analysis revealed that GAL7 and GAL1 are separated by approximately 2 kbp and share a common promoter region. Although GAL7, GAL10, and GAL1 are clustered in this order in previously studied hemiascomycetous yeasts, GAL10 was not found between GAL7 and GAL1 in S. naganishii. Southern blotting of S. naganishii chromosomal DNA showed that both the GAL7–GAL1 region and GAL10 are located on chromosome XI, but that GAL10 is located more than 10 kbp away from GAL7–GAL1. Thus, S. naganishii and S. cerevisiae, while related phylogenetically, do not share the same orientation with respect to GAL genes. These data are highly relevant to studies of chromosomal evolution in yeast.     

Genetic polymorphism of the swine major histocompatibility complex (SLA) class I genes, SLA-1, -2 and -3

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.The DNA sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank nucleotide databases and have been assigned the accession numbers, AB105379, AB105380, AB105381, AB105382, AB105383, AB105384, AB105385, AB105386, AB105388, AB105389, AB105390 and AB105391     

Proliferating cells in the rat anterior pituitary during the postnatal period: immunoelectron microscopic observations using monoclonal anti-bromodeoxyuridine antibody

Proliferating cells in the male rat anterior pituitary at 1, 3, 5, and 8 weeks of age were labeled with bromodeoxyuridine (BrdU) and studied by light and electron microscopic immunocytochemistry using anti-BrdU. They decreased in number from 402±31/mm² at 1 week to 50±1.5/mm² at 8 weeks, while their cell area increased by about twofold during this period. They had a slightly higher nucleus/whole cell (N/C) ratio than non-proliferating cells. According to their ultrastructure we classified them into granular and agranular cells. The percentage of granular cells ranged from 73% to 82% of all the proliferating cells during the period studied. They had many granules of various sizes and shapes, and some contained growth hormone and prolactin. Agranular cells, constituting 18–27% of proliferating cells, were small and had a high N/C ratio, indicating their immaturity. Moreover, they showed several features of folliculo-stellate (FS) cells: they showed no secretory granules in the cytoplasm, extended thin cytoplasmic processes, and sometimes they constructed a follicle among them. These results suggest: (1) the majority of proliferating cells were mature cells producing anterior pituitary hormone(s) and (2) most of the agranular proliferating cells maybe FS cells. The possibility of the latter is discussed.     

Carbonyl carbon transverse relaxation dispersion measurements and ms-μs timescale motion in a protein hydrogen bond network

A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R(2), dispersion experiment for carbonyl carbons was designed and executed to detect micros-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C(alpha)-C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly (13)C enriched proteins, unless care is taken to minimize the perturbation of the C(alpha) magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the C(alpha) region of the spectrum) pulses were employed in order to minimize C' signal modulation by C(alpha)-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [(13)C,(15)N, (2)H] ((2)H at non-labile hydrogen sites) labeled, and (2) uniformly (15)N/selectively-(13)C' labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly (13)C/(15)N/(2)H labeled sample was well suited to measure (15)N and (1)H R(2) dispersion as well as (13)C' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Thymocyte apoptosis induced by phosphorylation of histones is associated with the change in chromatin structure to allow easy accessibility of DNase

The inhibitors of protein phosphatase such as calyculin A and okadaic acid induce the apoptotic cell death in rat thymocytes. To clarify the molecular mechanism of these inhibitor-induced apoptosis, the effect of calyculin A on DNA fragmentation in the isolated nuclei were studied. A significant increase in DNA fragmentation was observed in the nuclei prepared from the cells treated with calyculin A that caused histone hyperphosphorylation. No changes of the activities of caspase-8 and -3 were observed in the extract from the cells treated with calyculin A. The circular dichroism analysis of soluble chromatin from calyculin A-treated thymocyte nuclei indicated that phosphorylation of histones decreased its alpha-helical content. Thus, the change in the chromatin structure may be due to the chemical modification of histones. Moreover, the structural change in chromatin preceded DNA fragmentation in the nuclei. Therefore, these results suggest that the change of chromatin structure allow easy accessibility of nuclear DNase to chromosomal DNA.     

Down's Syndrome: Up-Regulation of β-Amyloid Protein Precursor and τ mRNAs and Their Defective Coordination

Abstract: Almost all patients >40 years of age with Down's syndrome (DS) develop the pathology characteristic of Alzheimer's disease: abundant β-amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of β-amyloid protein precursor (APR) and τ in DS and age-matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up-regulation was not observed in two other neuronal proteins. A correlation between total APP and τ mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP-695 mRNA and three-repeat τ mRNA in DS, the proportionality between APP-751 mRNA and four-repeat τ mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up-regulation of τ mRNA as well as APP mRNA and disruption of the coordinate expression between APP-751 and four-repeat τ.     

β-Amyloid Protein Precursor and τ mRNA Levels Versus β-Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β-amyloid protein precursor (APP) and τ mRNAs are related to the formation of β-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto-chemically for β-amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β-amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β-protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β-amyloid and/or tangle formation in the aged human brain.