A novel type of sequence variation: multiple-nucleotide length polymorphisms discovered in the bovine genome

Three types of sequence variations--single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), and short tandem repeats (STRs)--have been extensively reported in mammalian genomes. In this study, we discovered a novel type of sequence variation, i.e., multiple-nucleotide length polymorphisms (MNLPs) in bovine UCN3 (Urocortin 3) and its receptor CRHR2 (corticotropin-releasing hormone receptor 2) genes. Both MNLPs featured involvement of multiple-nucleotide length polymorphisms (5-18 bases), low sequence identity, and 1.7- to 11-fold changes in promoter activity between two alleles. Therefore, this novel genetic complexity would contribute significantly to the evolutionary, functional, and phenotypic complexity of genomes within or among species.     

AN EPIZOOTIC OF FLORIDA MANATEES ASSOCIATED WITH A DINOFLAGELLATE BLOOM

Over a 10-wk period in early 1982, 39 Florida manatees ( Trichechus manatus latirostris ) were found dead in the lower Caloosahatchee River and nearby waters of southwestern Florida. Two were killed by boats. The remainder showed no evidence of trauma. Lesions indicative of infectious agents were not identified, and bacteriological and contaminant residue findings were unremarkable. Nonspecific lesions of congestion and hemorrhage were identified in brain tissue. Numerous reports were also received of manatee morbidity. Some distressed manatees showed no biochemical lesions in clinical analyses of blood samples and recovered quickly. Timing of manatee illnesses coincided with fish and double-crested cormorant ( Phalacrocorax auritus ) mortality and morbidity. A widespread bloom of the dinoflagellate red tide organism ( Gymnodinium breve ) also coincided with these incidents. G. breve produces potent neurotoxins (brevetoxins). Circumstantial evidence links these events, and possible routes of exposure may include ingestion of filter-feeding ascidians. Ecological conditions that magnified the extent of the epizootic included an early dispersal of manatees into the area from a nearby winter aggregation site and unusually high salinities that facilitated the inshore spread of the red tide bloom. Management responses to future episodes of red tide in manatee areas are suggested.     

DISTRIBUTION PATTERNS OF INDIVIDUALLY IDENTIFIABLE WEST INDIAN MANATEES (TRICHECHUS MANATUS) IN FLORIDA

Photographs of distinctively scarred manatees ( Trichechus manatus ) were taken at aggregation sites throughout Florida and assembled into a catalog for identifying individuals. Resightings of known manatees in different years or at different locations enabled us to document site fidelity and long-distance movements. Of the 891 individuals included in the catalog as of August 1986, 470 (53%) were resighted at least once. We documented 219 cases of seasonal returns to specific aggregation sites and 98 instances of movements between areas. Movements in excess of 820 km were documented for individuals on the east coast of Florida. Rapid movements south during early winter, as well as northerly spring movements, verify a seasonal migration pattern for many individuals along Florida's east coast. The wide-ranging migratory habits of manatees in Florida must be considered if meaningful management strategies are to be developed and implemented.     

Random DNA fragmentation allows detection of single-copy,single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.     

Molecular cloning and characterization of a leukemia-inducing myeloproliferative sarcoma virus and two of its temperature-sensitive mutants.

The myeloproliferative sarcoma virus (MPSV) induces extensive hematopoietic changes, including spleen foci in adult mice, and transforms fibroblasts in vitro. NRK nonproducer cell lines of MPSV and ts temperature-sensitive mutants were analyzed by restriction enzyme digestion and Southern blotting. EcoRI fragments containing the proviral DNAs of MPSV and two temperature-sensitive mutants and rat cellular sequences homologous to c-mos were molecularly cloned. By comparing restriction enzyme cleavage sites, it was shown that the MPSV genome consists only of sequences related either to Moloney murine leukemia virus or to the c-mos mouse oncogenic sequences. Two regions of fragment heterogeneity were observed: (i) in the defective pol gene, where MPSV and the two cloned temperature-sensitive mutants were different from Moloney murine sarcoma virus and from each other, although MPSV wild-type retained more of the pol gene than any of the Moloney murine sarcoma virus isolates; (ii) in the area 3' to the mos gene, which was identical in MPSV and its temperature-sensitive mutants but different from other Moloney murine sarcoma virus variants. Transfection of cloned MPSV DNA in RAT4 cells and virus rescue on infection with Friend murine leukemia virus yielded MPSV which transformed fibroblasts in vitro and also induced spleen foci in adult mice, thus proving that both properties are coded by the same viral genome.     

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert ³H-labeled inulin or the degradable ¹²⁵I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4°C were effectively internalized and processed intracellulary after shifting the temperature to 37°C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37°C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37°C. From the temperature-change experiments we conclude that, at 4°C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37°C.     

Nitrate and Nitrite Reduction by Wolffia arrhiza

Nitrate reductase was not found to be present in or associated with partially purified, intact chloroplasts aqueously isolated from Wolffia arrhiza. Such chloroplasts are capable of using nitrite but not nitrate as an electron acceptor during light-stimulated electron transport in the absence of additional cytoplasmic components. When nitrite acts as an electron acceptor under these conditions, on the average 1.5 moles of oxygen are evolved per mole of nitrite reduced by the chloroplasts, indicating a probable reduction of nitrite to ammonia. Chloroplasts ruptured by osmotic shock fail to reduce nitrite in the absence of additional components.     

Oxygen evolution and the permeability of the outer envelope of isolated whole chloroplasts

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.

Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.

A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.

     

Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.