Linkage analysis of the murine interferon-alpha locus on chromosome 4.

Southern blot analysis with a murine interferon-alpha2 (MuIFN-alpha2) cDNA probe revealed restriction fragment polymorphism of EcoRI- and HindIII-digested C57BL/6 and BALB/cDNA. The inheritance pattern of this polymorphism was examined using DNA from each of the seven recombinant inbred strains derived from C57BL/6 and BALB/c; the strain distribution pattern suggests linkage of INF-alpha genes to two histocompatibility loci on chromosome 4. Southern blot analysis of DNA from six bilinear congenic strains carrying different fragments of the BALB/c chromosome 4 on a C57BL/6 background showed linkage of IFN-alpha genes to the histocompatibility locus H-15. It can therefore be concluded that the IFN-alpha gene cluster is situated on chromosome 4 near the H-15 locus, between loci Mup-1 and b.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice.

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.

Heterogeneity in mantle carbon content from CO2-undersaturated basalts.

The amount of carbon present in Earth's mantle affects the dynamics of melting, volcanic eruption style and the evolution of Earth's atmosphere via planetary outgassing. Mantle carbon concentrations are difficult to quantify because most magmas are strongly degassed upon eruption. Here we report undegassed carbon concentrations from a new set of olivine-hosted melt inclusions from the Mid-Atlantic Ridge. We use the correlations of CO2 with trace elements to define an average carbon abundance for the upper mantle. Our results indicate that the upper mantle carbon content is highly heterogeneous, varying by almost two orders of magnitude globally, with the potential to produce large geographic variations in melt fraction below the volatile-free solidus. Such heterogeneity will manifest as variations in the depths at which melt becomes interconnected and detectable, the CO2 fluxes at mid-ocean ridges, the depth of the lithosphere-asthenosphere boundary, and mantle conductivity.

The evolution of lipophilin genes from invertebrates to tetrapods: DM-20 cannot replace proteolipid protein in CNS myelin.

The proteolipid protein (PLP) gene encodes two myelin-specific protein isoforms, DM-20 and PLP, which are members of the highly conserved lipophilin family of transmembrane proteins. While the functions of this family are poorly understood, the fact that null mutations of the PLP gene cause leukodystrophy in man is testament to the importance of DM-20 and PLP in normal CNS function. PLP differs from DM-20 by the presence of a 35 amino acid domain exposed to the cytoplasm, which is not encoded by other lipophilin genes and appears to have arisen in amphibians approximately 300 million years before present. However, the lipophilin gene family can be traced back at least 550 million years and is represented in Drosophila and silkworms. Thus, from an evolutionary perspective PLP can reasonably be anticipated to perform functions in CNS myelin that cannot be accomplished by other lipophilins. Herein we use a novel knock-in strategy to generate mice expressing wild-type levels of a Plp gene that has been modified to encode only DM-20. Although DM-20 is incorporated into functional compact myelin sheaths in young animals, our data show that the 35 amino acid PLP-specific peptide is required to engender the normal myelin period and to confer long-term stability on this multilamellar membrane.

Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Rearrangement of genes located on homologous chromosomal segments in mouse and man: the location of genes for alpha- and beta-interferon, alpha-1 acid glycoprotein-1 and -2, and aminolevulinate dehydratase on mouse chromosome 4.

Gene mapping studies to determine the order of alpha- and beta-interferon (Ifa, Ifb), aminolevulinate dehydratase (Lv), and alpha-1 acid glycoprotein-1 and -2 (Orm-1, Orm-2) relative to each other and to the reference genes brown (b), B-cell maturation factor responsiveness (Bmfr-1), and major urinary protein-1 (Mup-1) are reported. The most likely order was Mup-1--Lv--b--Orm-1, Orm-2--Ifa, Ifb--Bmfr-1. This order suggested that two chromosomal segments located on chromosome 4 in the mouse and chromosome 9 in man have been conserved since divergence of lineages leading to man and mouse; these segments are marked by soluble aconitase-1 (Aco-1) and galactose-1 phosphate uridyl transferase (Galt) and by Lv and Orm-1. This order also demonstrated that, although genes located on opposite arms of chromosome 9 in man remain syntenic in the mouse, gene order has not been conserved; Ifa and Ifb are not located in their expected locations near Aco-1 and Galt. The position of Ifa and Ifb between Orm-1 and Bmfr-1 could not be determined with certainty because of apparent heterogeneity in recombination frequencies between crosses involving conventional laboratory strains of mice and crosses involving interspecific matings between laboratory mice and Mus spretus. This result suggests that caution must be exercised when using M. spretus in linkage crosses.

Synthesis of fusion and mature murine alpha interferons in Escherichia coli.

Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.

Mapping of murine interferon-alpha genes to chromosome 4.

A cDNA library was constructed from polysomal poly(A)+RNA from Newcastle disease virus (NDV)-induced mouse C243 cells, and screened with a human interferon-alpha (HuIFN-alpha) cDNA probe. A cDNA clone for one of the murine interferon-alpha (MuIFN-alpha) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN-alpha 2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot analysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-alpha gene family. The MuIFN-alpha genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs.

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye.

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.

Testing the role of the cell-surface molecule Thy-1 in regeneration and plasticity of connectivity in the CNS.

Thy-1 is a cell-surface signaling molecule of the Ig superfamily implicated in the regulation of neurite outgrowth, synaptic function and plasticity. There is, however, no consensus as to its precise function in the nervous system, and it remains unclear or untested as to what its role is in the development, maintenance and plasticity of neuronal connectivity in the intact brain and whether it is essential for any of the purported functions which have been attributed to it based largely on in vitro bioassays. Here, we have engineered transgenic mice with a targeted deletion of the Thy-1 gene and, after characterizing the development of their corticospinal and thalamocortical pathways, subjected them at adulthood to paradigms of axonal regeneration and plasticity which can be readily induced during development. Quantitative analyses of the brains and spinal cords of adult null mutants showed normal cellular organization, normal anatomical features of the corticospinal and thalamocortical pathways, and basic neurophysiological properties of thalamocortical synaptic transmission which were quantitatively indistinguishable from wild-type mice. Despite the absence of Thy-1, corticospinal axons in adult mutants failed to exhibit overt regeneration following spinal cord lesion; likewise, the terminal arbors of ventrobasal thalamocortical axons also failed to reorganize in adult barrel cortex in response to whisker cautery, although they did so during a developmental critical period identical to that displayed by wild-type mice.Taken together, these results suggest that Thy-1 is not essential for the normal development and maintenance of major axon pathways and functional synaptic connections, nor would it appear to be critically important for inhibiting or promoting axonal growth, regeneration and plasticity in the developing and mature CNS.

Expression of Thy-1/lacZ fusion genes in the CNS of transgenic mice.

Thy-1 is a cell surface glycoprotein of unknown function that is found on nerve cells and mature T-lymphocytes. To study the regulation of Thy-1 gene expression, mouse Thy-1.2 genomic sequences were joined to various marker sequences and the resulting chimeric constructs were used to produce nearly three dozen independent lines of transgenic mice. The starting point for our studies was an 8.2 kb EcoRI fragment that begins 1.7 kb 5' to the transcription start site and ends with 1.3 kb of 3' flanking sequences. Addition of a small marker oligonucleotide to the 3' untranslated region of this fragment had little or no effect on gene regulation. All of the lines derived from injection of this construct expressed the transgene in the appropriate tissues. Thus, as expected, the Thy-1.2 genomic fragment contains all of the information necessary for tissue-specific, position-independent expression of the modified transgene. Unexpectedly, Thy-1/lacZ hybrid genes did not mimic this behavior. Using either mRNA or histochemical detection of lacZ protein, these constructs were expressed in patterns that varied dramatically from line to line. This behavior suggests that integration site-specific effects dominate the cis-active Thy-1 regulatory elements leading to wide variability of expression. This is further emphasized by the observation that the bacterial reporter protein was found in a few non-neuronal cell-types, in contrast to the known pattern of native Thy-1 expression. These results suggest that either the Thy-1.2 sequences which are necessary for appropriate brain-specific expression are not contained solely within the proposed CNS enhancer in the first intron, or that fusion of the Thy-1.2 sequences with the lacZ coding region may disrupt normal Thy-1 regulatory signals (or result in the creation of new regulatory elements).

Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish.

Gastrointestinal function depends upon coordinated contractions to mix and propel food through the gut. Deregulation of these contractions leads to alterations in the speed of material transit through the gut, with potentially significant consequences. We have developed a method for visualizing intestinal transit, the physiological result of peristaltic contractions, in larval zebrafish. This method allows direct, non-invasive observation of luminal content as it traverses the gut. Using this method, we characterized gastrointestinal transit in zebrafish larvae at 7 days postfertilization. In addition, we used this transit assay to assess the physiological consequences of reduced or absent enteric neurones on intestinal transit in larval zebrafish. This may facilitate the use of the zebrafish for investigating the effect of compounds and candidate genes on gastrointestinal motility.

Very early detection of changes associated with cellular activation using a modified flow cytometer.

A sample station modification previously described has been redesigned to provide greater flexibility and enhanced performance. The improved modification provides mixing and temperature regulation in a compact unit that mounts close to the nozzle holder for reduced transit times, allowing for addition of mediators to a sample in place on the flow cytometer, with observation of results in approximately 1 s. An electronic circuit activated at the time of injection generates full-scale pulses in the forward scatter channel. This provides the data with a time stamp for direct correlation of injection and cellular response. A detailed description of the modification, performance verification data, and practical applications in the measurements associated with cellular activation are presented.

Sample station modification providing on-line reagent addition and reduced sample transit time for flow cytometers.

A flow cytometer was equipped with a modified sample station to facilitate on-line addition of mediators to the sample and reduce the time of delivery of the sample to the interrogation point. The ready availability of materials and straightforward nature of this design make modifications simple and facilitate measurements of cellular activation. Parameters such as pH, membrane fluidity, and calcium mobilization are easily measured in this system, because detection can be made less than 4 s after addition of mediator with no interruption of sample flow. The sample station modification is described in detail along with methods for mixing and temperature control.

Characterization of a mouse interferon gene locus I. Isolation of a cluster of four alpha interferon genes.

A BALB/c mouse genomic library was screened with a murine interferon alpha 2 (MuIFN-alpha 2) cDNA coding region fragment. Eight clones were isolated which contain different mouse chromosomal segments related to the MuIFN-alpha 2 probe and a 28 kilobase (kb) region of mouse genomic DNA containing four different MuIFN-alpha genes (alpha 1, alpha 4, alpha 5 and alpha 6) was identified and characterized; an intergenic 1000 nucleotide long conserved sequence was found to be associated with three of these four alpha genes, indicating that this alpha-IFN gene cluster evolved through tandem duplications. Sequence analysis revealed the absence of a polyadenylation site in the 3' untranslated region of MuIFN-alpha 1, and showed that one of the genes (alpha 4) contains an internal deletion of 5 amino acids in the coding region.

Characterization of a mouse interferon gene locus II. Differential expression of alpha-interferon genes.

A cluster of four MuIFN-alpha genes was recently isolated and characterized (1); one of the genes in this cluster had, in the coding region, an internal deletion of 5 amino acids. Bacterial expression plasmids were constructed to examine the effect of this deletion on the antiviral activity of the MuIFN-alpha 4 peptide and it was found that the alpha 4 interferon peptide had a 100-fold lower antiviral activity than full length alpha-interferon proteins when expressed in E. coli. Three of the four MuIFN-alpha genes identified were expressed coordinately in L-cells infected with NDV. The relative levels of alpha 4 mRNA were substantially higher than the levels of the other alpha mRNAs. Comparison of the 5' end flanking sequences of these four alpha interferon genes revealed that the promoter sequences of alpha 1, alpha 5 and alpha 6 are more homologous to each other than to the alpha 4 promoter which also contains a G rich cluster not seen in the other three promoters.

Differential effect of poly rI.rC and Newcastle disease virus on the expression of interferon and cellular genes in mouse cells.

The expression of type I murine interferon (MuIFN) genes and several other cellular genes was examined in poly rI.rC induced and Newcastle disease virus (NDV) infected mouse cells. Northern analysis of RNA from induced L cells revealed that the MuIFN-alpha s are expressed efficiently in NDV infected cells but only at low levels in poly rI.rC induced cells. MuIFN-beta 1, however, is expressed equally well in cells treated with poly rI.rC or infected with NDV. As shown by the use of a probe specific for poly rI.rC, interferon induction correlates with the cellular uptake of poly rI.rC into the cells. The relative levels of alpha and beta 1 mRNAs in the cells reached a maximum at 10 hr after the induction which indicates coordinate expression of alpha and beta 1 interferon genes. The effect of viral infection on the expression of two murine genes coinduced with interferon (pMIF20/11 and pMIF3/10) and several cellular genes was also examined. While pMIF20/11 is an inducible gene, the pMIF3/10 gene is expressed constitutively in mouse L cells. Viral infection, but not poly rI.rC treatment, enhanced the expression of the pMIF3/10 gene, as well as two other cellular genes; H-2 and c-myc, however, the expression of beta-actin gene was unaltered. These data indicate that enhancement of gene expression in virus infected cells in not limited to the interferon system.

Practical considerations for the selection and use of optical filters in flow cytometry.

The selection of proper optical filters for various excitation and emission requirements is critical in flow cytometry. Problems which arise in the selection and utilization of optical filters, and the solutions to these problems, are the subject of this article.