Polymorphism and Gene Conversion in Mouse α-Globin Haplotypes

We have cloned and characterized three distinct alpha-globin haplotypes obtained from inbred strains of the mouse, Mus domesticus. We report here the complete nucleotide sequence of the six alpha-globin genes that the haplotypes contain. Our analysis of these genes and those from one other previously described haplotype indicates that recurrent gene conversion events have played a major role in their history. The pattern of nucleotide substitutions suggests that conversions have occurred both within and between haplotypes. Limited segments of coding and noncoding DNA have been involved in these gene conversion events. In two of the haplotypes, the nonallelic genes of each maintain DNA sequence identity over discrete intervals and encode the same alpha-globin polypeptide. On the other hand, the coding regions of some genes have accumulated replacement changes that result in distinct alpha-globins. In one instance, these changes appear to reflect positive selection of advantageous mutations.     

A novel fibrillar structure in cultured cells detected by a monoclonal antibody

Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.     

Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome.

Bacteriophage HP1c1 lysogenizes its host Haemophilus influenzae Rd by inserting its genome into the bacterial chromosome. The DNA segments corresponding to the integration regions on the phage and host chromosomes and the two junctions formed between phage and host sequences on lysogenic insertion were isolated and propagated in Escherichia coli HB101 as hybrid plasmids by using pBR322 as the vector. The nucleotide sequences in the vicinity of the point of recombinational insertion were determined. Phage and host DNA shared an extensive, nearly identical, segment that was 183 base pairs long. This segment consisted of 93 identical residues and a 27-residue portion containing 6 mismatches, followed by 63 identical residues. Recombinational insertion occurred within the 63-residue identical segment and involved neither duplication nor deletion of any residues. Short inverted repeats consisting of clustered A-T base pairs were present within the two 27-residue segments. Two additional sites on the host chromosome showed significant hybridization to the phage-host homology region.     

Structural and dynamic properties of a fluorouracil-adenine base pair in DNA studied by proton NMR

An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.     

Studies on the metabolism of retinol-binding protein by primary hepatocytes from retinol-deficient rats

Studies were conducted to explore the regulation of retinol-binding protein (RBP) metabolism in cultured primary hepatocytes from retinol-deficient rats. Newly isolated hepatocytes from retinol-deficient rats contained elevated levels (3.4-fold) of RBP, compared to hepatocytes from normal (retinol-adequate) rats. Addition of retinol to retinol-depleted hepatocytes stimulated RBP secretion by the cells in a concentration-dependent manner. Maximal stimulation of RBP secretion was seen with a retinol level of 0.3 micrograms/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol-deficient hepatocytes. Addition of dexamethasone to retinol-deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol-depleted cells differed dramatically from effects seen in other studies with normal (retinol-adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.     

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

In situ detection of early replication phases of a gemini virus in legume protoplasts

Protoplasts of Phaseolus vulgaris L. (Top Crop), infected with bean golden mosaic virus, were isolated and fixed by various methods for in situ hybridization. An iodine-125 labeled probe was made from the replicative form of the virus. The localization and quantitation was done by autoradiography. Cell wall removal lowered the background and allowed a more accurate analysis. RNase was used to eliminate the possibility of hybrids to RNA. The evidence suggests a sequence of virus movements starting from rough endoplasm reticulum, moving to the nuclear membrane, and finally with the highest concentration inside the nucleus.Abbreviations BGMV bean golden mosaic virus - rfBGMV or rfDNA replicative double-stranded DNA virus - ssDNA single-stranded virus     

Structural and dynamic properties of a bromouracil-adenine base pair in DNA studied by proton NMR

We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.     

ASSESSING NORTHERN ELEPHANT SEAL FEEDING HABITS BY STOMACH LAVAGE

Stomach lavaging was used to study the feeding habits of northern elephant seals ( Mirounga angustirostris ) found on San Miguel Island, California, during the spring of 1984. Fifty-nine elephant seals were chemically immobilized with an intramuscular injection of ketamine hydrochloride. Once immobilized, an animal's stomach was intubated, filled with 3–4 liters of water to create a slurry of the undigested food items, and evacuated into a collection device. The stomachs of 57 (96.6%) of the animals lavaged contained identifiable parts of prey. Twenty-nine different food items were identified, 12 of which have not been previously reported as prey of the northern elephant seal: two teleost fish, Coryphaenoides acrolepis (Pacific rattail) and another unidentified macrourid; two crustaceans, Pasiphaea pacifica (glass shrimp) and Euphausia sp.; six squid, Abraliopsis felis, Gonatus berryi, Histioteuthis dofleini, Cranchia scabra, Taonius pavo, and Galiteuthis sp. and two octopi, Octopus dofleini and Octopus rubescens.