CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae.

A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions. Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups. The CIK1 gene was cloned and characterized. Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain. Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells. Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus. Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type. Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth. Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment. These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.     

Angiotensin converting enzyme immunohistochemistry in rat brain and pituitary gland: correlation of isozyme type with cellular localization

We have localized angiotensin converting enzyme in rat brain and pituitary gland immunohistochemically with an anti-rat lung angiotensin converting enzyme monoclonal antibody. The distribution of immunoreactive angiotensin converting enzyme is identical with that of binding sites for the angiotensin converting enzyme inhibitor, [3H]captopril. Most intense staining is in the choroid plexus and subfornical organ, with intermediate values in the caudate-putamen, globus pallidus, entopeduncular nucleus, pars reticulata of the substantia nigra, posterior pituitary and anterior pituitary. Lower levels are observed in the supraoptic and paraventricular nuclei of the hypothalamus. Within the basal ganglia angiotensin converting enzyme immunoreactivity is distributed throughout the neuropil; no cell bodies are stained, even after colchicine treatment. The punctate pattern of immunoreactivity in the anterior pituitary corresponds to the distribution of endothelial cells. The posterior pituitary is stained diffusely. Angiotensin converting enzyme is increased by 45% in the posterior lobe after pituitary stalk section, demonstrating that this diffuse staining is associated with pituicytes. Antibody specificity was demonstrated by the immunoaffinity purification of angiotensin converting enzyme to homogeneity from crude tissue extracts using anti-angiotensin converting enzyme antibody and protein A-sepharose. The apparent molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis of lung, choroid plexus and anterior pituitary angiotensin converting enzyme is 175,000. In the substantia nigra and caudate putamen, where angiotensin converting enzyme is localized to neuronal as opposed to epithelial cells, the molecular weight is 165,000. The pituicyte angiotensin converting enzyme of the posterior pituitary is 170,000 daltons.     

The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys

The nonstructural (NS) genes of avian influenza A viruses have been divided into two groups on the basis of nucleotide sequence homology, which we have referred to here as alleles A and B. We sequenced the NS genes of eight additional avian influenza A viruses in order to define the differences between these two alleles more thoroughly. Four of the viruses had NS gene sequences which resembled that of A/FPV/Rostock/34 and belonged to allele A while the other four viruses had NS gene sequences more similar to that of A/Duck/Alberta/76 and belonged to allele B. There was approximately 90% sequence homology within alleles and 72% homology between alleles. As previously reported the NS genes of human influenza A viruses belong to allele A. We constructed single gene avian-human reassortant influenza A viruses containing an allele A or B NS gene segment from an avian influenza A virus and all other genes from a human influenza A virus and tested these reassortants for their ability to grow in the respiratory tract of a nonhuman primate. Reassortants containing an avian NS gene segment of allele B were significantly restricted in growth in the respiratory tract of squirrel monkeys while reassortants with an allele A NS gene segment were not. The divergent evolution of the B NS allele in birds may have resulted in gene products which do not function optimally in cooperation with genes from a human virus in viral replication in primate respiratory epithelium.     

Seven novel mutations in the adenosine deaminase (ADA) gene in patients with severe and delayed onset combined immunodeficiency: G74C,V129M,G140E,R149W,Q199P, 462delG,and E337del

The degree of immunodeficiency associated with deficiency of adenosine deaminase (ADA) is variable. Most patients are infants with severe combined immunodeficiency (SCID), but in about 20 percent immune dysfunction becomes manifest later in childhood ("delayed-onset"); several patients with "late" or "adult" onset of immune dysfunction have been diagnosed at 15–39 years. Over 40 ADA gene mutations have thus far been identified. To better define the genotype-phenotype relationship, we report 7 novel ADA mutations, including 5 missense mutations (G74C, V129M, G140E, R149W, Q199P) and two short deletions (462delG, E337del). These were identifed among 7 patients (3 with SCID and 4 with delayed-onset). A homozygote for 462delG had SCID, whereas patients homozygous or heterozygous for V129M had delayed-onset. Two other delayed-onset patients, one heterozygous for G74C and the other for Q199P, each had a second allele carrying the previously reported "severe" mutation G216R. These findings are consistent with previous observations suggesting that, in general, SCID occurs when both alleles eliminate ADA function, and a milder phenotype when at least one allele can supply a low level of function. Hum Mutat 11:482, 1998. © 1998 Wiley-Liss, Inc.     

Porphyrin Loading of Lipofuscin Granules in Inflamed Striated Muscle

To further the understanding of oxidative effects on inflammation injury to muscle fiber structure, fluorescent imaging analysis of human striated muscle tissues from a variety of inflammatory or postinflammatory etiologies was undertaken in a search for accumulated coproporphyrin, a red autofluorescent byproduct of heme biosynthesis that would theoretically be formed under oxidative insult. Using a differential excitation method of in situ analysis, porphyrin autofluorescence was detected in intact fibers within the context of the yellow autofluorescent subsarcolemmal lipofuscin granules. Relative measurements of porphyrin concentration in the granules from different patients indicated that the acute/subacute inflammatory specimens grouped significantly higher than the more chronic inflammatory and nonpathological specimens. Myoglobin was also found to be associated with the granules. Myoglobin heme iron could potentially serve as a Fenton reagent for the intracellular generation of hydroxyl radicals, which are responsible for the oxidation of the porphyrinogens. High-performance liquid chromatography analysis of extracted dense particles revealed coproporphyrin as the sole porphyrin present. The observation of coproporphyrin within lipofuscin granules, previously unreported, suggests that lipofuscin accumulation in striated muscle may begin under conditions of acute oxidative stress, as marked by the oxidation of extramitochondrial porphyrinogens that are immediately incorporated into the granules.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.