In plants, N-linked glycans are processed in the Golgi apparatus to complex-type N-glycans of limited size containing a beta(1,2)-xylose and/or an alpha(1,3)-fucose residue. Larger mono- and bi-antennary N-linked complex glycans have not often been described. This study has re-examined the structure of such plant N-linked glycans, and, through both immunological and structural data, it is shown that the antennae are composed of Lewis a (Le(a)) antigens, comprising the carbohydrate sequence Gal beta 1-3[Fuc alpha 1-4]GlcNAc. Furthermore, a fucosyltransferase activity involved in the biosynthesis of this antigen was detected in sycamore cells. This is the first characterization in plants of a Lewis antigen that is usually found on cell-surface glycoconjugates in mammals and involved in recognition and adhesion processes. Le(a)-containing N-linked glycans are widely distributed in plants and highly expressed at the cell surface, which may suggest a putative function in cell/cell communication.

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh. Entamoeba histolytica infection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child of E. histolytica-associated diarrhea and 0.03 episodes/child of E. histolytica-associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.

Galectin-3 (gal-3), a pleiotrophic protein, is an important regulator of tumor metastasis, which like beta-catenin shuttles between the nucleus and the cytosol in a phosphorylation-dependent manner. We report herein that beta-catenin stimulation of cyclin D1 and c-myc expression is gal-3 dependent. Gal-3 binds to beta-catenin/Tcf complex, colocalizes with beta-catenin in the nucleus, and induces the transcriptional activity of Tcf-4 as determined by the TOP/FOPFLASH reporter system. We have identified the beta-catenin-gal-3-binding sequences, which are in the NH2 and COOH termini of the proteins encompassing amino acid residues 1 to 131 and 143 to 250, respectively. These data indicate that gal-3 is a novel binding partner for beta-catenin involved in the regulation of Wnt/beta-catenin signaling pathway.

The neuropeptide galanin coexists with norepinephrine and serotonin in neural systems mediating emotion. Previous findings suggested that galanin modulates anxiety-related behaviors in rodents. Three galanin receptor subtypes have been cloned; however, understanding their functions has been limited by the lack of galanin receptor subtype-selective ligands. To study the role of the galanin GAL-R1 receptor subtype in mediating anxiety-related behavior, we generated mice with a null mutation in the Galr1 gene. GAL-R1 -/- are viable and show no abnormalities in health, neurological reflexes, motoric functions, or sensory abilities. On a battery of tests for anxiety-like behavior, GAL-R1 -/- showed increased anxiety-like behavior on the elevated plus-maze test. Anxiety-related behaviors on the light/dark exploration, emergence, and open field tests were normal in GAL-R1 -/-. This test-specific anxiety-like phenotype was confirmed in a second, independent cohort of GAL-R1 null mutant mice and +/+ controls. Principal components factor analysis of behavioral scores from 279 mice suggested that anxiety-like behavior on the elevated plus-maze was qualitatively distinct from behavior on other tests in the battery. In addition, exposure to the elevated plus-maze produced a significantly greater neuroendocrine response than exposure to the light/dark exploration test, as analyzed in normal C57BL/6J mice. These behavioral findings in the first galanin receptor null mutant mouse are consistent with the hypothesis that galanin exerts anxiolytic actions via the GAL-R1 receptor under conditions of relatively high stress.

The glycosylation enzyme alpha-1,3-galactosyltransferase (alpha 1,3GT; UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase, EC 2.4.1.151) displays a unique pattern of distribution in mammals. It synthesizes an abundance of Gal(alpha 1-3)Gal(beta 1-4)GlcNAc-R (alpha-galactosyl) epitopes within the Golgi apparatus of cells of nonprimate mammals, prosimians, and New World monkeys (platyrrhines). The catarrhines, which include Old World monkeys, apes, and humans, lack this enzyme activity because of the inactivation of the alpha 1,3GT gene. In contrast, the catarrhines produce large amounts of antibodies, designated anti-Gal, against the alpha-galactosyl epitope. The inactivation of the alpha 1,3GT gene in ancestral catarrhines was probably the result of an intensive evolutionary pressure for alteration in the makeup of cell surface carbohydrates (i.e., suppression of alpha-galactosyl epitope expression) and for the production of the anti-Gal antibody. To determine the period in which the alpha 1,3GT gene was inactivated in ancestral catarrhines, comparative sequencing of a 370-base-pair region of this gene was performed by polymerase chain reactions with DNA of various primates. The data suggest that alpha 1,3GT inactivation occurred rather late in the course of catarrhine evolution (less than 28 million years ago), as separate events in apes and in Old World monkeys, after the two groups diverged from each other.

We have isolated a new yeast gene (PCC1) that codes for a factor homologous to human cancer-testis antigens. We provide evidence that Pcc1p is a new transcription factor and that its mutation affects expression of several genes, some of which are involved in cell cycle progression and polarized growth. Mutation of Pcc1p also affects the expression of GAL genes by impairing the recruitment of the SAGA and Mediator co-activators. We characterize a new complex that contains Pcc1p, a kinase, Bud32p, a putative endopeptidase, Kae1p and two additional proteins encoded by ORFs YJL184w and YMLO36w. Genetic and physical interactions among these proteins strongly suggest that this complex is a functional unit. Chromatin immunoprecipitation experiments and multiple genetic interactions of pcc1 mutants with mutants of the transcription apparatus and chromatin modifying enzymes underscore the direct role of the complex in transcription. Functional complementation experiments indicate that the transcriptional function of this set of genes is conserved throughout evolution.

The promoters of the highly expressed and stringently regulated GAL genes of Saccharomyces cerevisiae, are useful for expressing proteins in this organism. However, two problems complicate their use. First, because growth on glucose causes prolonged repression of GAL expression, cells are most rapidly induced after growth on nonfermentable carbon sources, conditions which usually support poor growth. Second, because the inducer of the GAL genes (galactose) also serves as a carbon source, the level of inducer is continually diminishing during growth of a Gal+ strain, which may lead to reduced GAL expression. To solve the first problem, we have employed strains that carry the reg1-501 mutation, which eliminates glucose repression of GAL expression. This gene has been shown to be located on the right arm of chromosome IV, distal but tightly linked to the TRP1 gene. We demonstrate that expression from GAL promoters is efficiently and rapidly induced in these reg1 strains by the addition of galactose to a culture growing in glucose medium. Levels of galactose as low as 0.02% can be used to obtain a 1500-fold induction of gene expression from GAL promoters in this strain. To surmount the second problem, we have used a gal1 mutant, deficient in the enzyme that catalyzes the first step of galactose utilization. We show that high levels of expression from GAL promoters are achieved rapidly in these mutants, for which galactose is a gratuitous inducer.(ABSTRACT TRUNCATED AT 250 WORDS)

CD69 is a membrane-bound, type II C-lectin receptor. It is a classical early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (γδ) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates some specific functions of selected T-cell subsets, determining the migration-retention ratio as well as the acquisition of effector or regulatory phenotypes. Specifically, CD69 regulates the differentiation of regulatory T (Treg) cells as well as the secretion of IFN-γ, IL-17, and IL-22. The identification of putative CD69 ligands, such as Galectin-1 (Gal-1), suggests that CD69-induced signaling can be regulated not only during cognate contacts between T cells and antigen-presenting cells in lymphoid organs, but also in the periphery, where cytokines and other metabolites control the final outcome of the immune response. Here, we will discuss new aspects of the molecular signaling mediated by CD69 and its involvement in the metabolic reprogramming regulating TH-effector lineages.

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.

The activation of human T-lymphocytes by anti-CD3 antibodies and interleukin-2 results in a marked increase in apparent molecular weight of the major cell-surface sialoglycoprotein. Both forms of the sialoglycoprotein were identified as leukosialin by a monospecific antiserum, and the differences in molecular weight were found to be due to changes in the carbohydrate structures. Our results suggest that resting T-lymphocytes express on leukosialin the disialotetrasaccharides NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)Gal-NAc-Ser/Thr, whereas activated human T-cells carry on leukosialin exclusively the more complex structures NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-Ser/Thr. The radical shift in the biosynthetic pathway of O-glycans in activated T-lymphocytes compared to resting cells is apparently caused by a decrease of alpha 2----6 sialyltransferase activity and by the parallel dramatic stimulation of the beta 1----6GlcNAc-transferase. Since both enzymes compete for the same precursor substrate, the coordinate changes in their activities are most likely responsible for the complete change of the carbohydrate structures on leukosialin during the activation of human T-lymphocytes.

The first and major clinical obstacle in xenotransplantation is antibody-mediated hyperacute rejection. Although human natural antibodies against Galalpha1,3Gal (Gal) antigens, which are common on porcine cells and organs, have been identified to play a major role in hyperacute rejection, other natural antibodies against non-Gal epitopes may be also involved in the process. Here, we present evidence suggesting that the majority of human anti-non-Gal antibodies are specific for carbohydrate structures carrying terminally linked N-glycolylneuraminic acid (NeuGc), a xenoantigen existing in almost all animals except humans. Furthermore, this anti-NeuGc activity is detectable in 85% of healthy humans, implicating the involvement of NeuGc in hyperacute rejection and the importance of developing strategies for removing NeuGc for clinical xenotransplantation.

COUP-TFII, an orphan member of the steroid receptor superfamily, has been implicated in mesenchymal-epithelial interaction during organogenesis. The generation of a lacZ knock-in allele in the COUP-TFII locus in mice allows us to use X-gal staining to follow the expression of COUP-TFII in the developing stomach. We found COUP-TFII is expressed in the mesenchyme and the epithelium of the developing stomach. Conditional ablation of floxed COUP-TFII by Nkx3-2Cre recombinase in the gastric mesenchyme results in dysmorphogenesis of the developing stomach manifested by major patterning defects in posteriorization and radial patterning. The epithelial outgrowth, the expansion of the circular smooth muscle layer and enteric neurons as well as the posteriorization of the stomach resemble phenotypes exhibited by inhibition of hedgehog signaling pathways. Using organ cultures and cyclopamine treatment, we showed downregulation of COUP-TFII level in the stomach, suggesting COUP-TFII as a target of hedgehog signaling in the stomach. Our results are consistent with a functional link between hedgehog proteins and COUP-TFII, factors that are vital for epithelial-mesenchymal interactions.

Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell-cell and cell-matrix interactions. UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase (GalT) (EC 2.4.1.38) is the enzyme which transfers galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) of complex-type N-glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell-cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT-deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT-deficient mice were born normally and were fertile, but they exhibited growth retardation and semi-lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.

Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1alpha (HIF-1alpha). To analyze EPAS1 domains responsible for transactivation and oxygen-regulated function, we constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain, plus or minus the VP16 activation domain. Two transactivation domains were defined in EPAS1; a C-terminal domain (amino acids 828-870), and a larger internal domain (amino acids 517-682). These activation domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity. Two types of activity were defined. Sequences lying N-terminal to and overlapping the internal transactivation domain conferred regulated repression on the VP16 transactivator. Sequences lying C-terminal to this internal domain conveyed repression and oxygen-regulated activity on the native EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion. Fusions containing internal but not C-terminal regulatory domains manifested regulation of fusion protein level. Comparison of EPAS1 with HIF-1alpha demonstrated a similar organization for both proteins, and for the C terminus defined a conserved RLL motif critical for inducibility. Overall, EPAS1 sequences were less inducible than those of HIF-1alpha, and inducibility was strikingly reduced as their expression level was increased. Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1alpha, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains.

Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure. Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling, and dysfunction. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a naturally occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and heart failure postmyocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction, and these effects are mediated by the transforming growth factor (TGF)-beta/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 wk: 1) vehicle (saline, n = 8); 2) Ac-SDKP (800 microg x kg(-1) x day(-1), n = 8); 3) Gal-3 (12 microg/day, n = 7); and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction, cardiac output, and transmitral velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF-beta expression and Smad3 phosphorylation by Western blot. We found that, in the left ventricle, Gal-3 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis, and causes cardiac hypertrophy; 2) increased TGF-beta expression and Smad3 phosphorylation; and 3) decreased negative change in pressure over time response to isoproterenol challenge, ratio of early left ventricular filling phase to atrial contraction phase, and left ventricular ejection fraction. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3-induced cardiac inflammation, fibrosis, hypertrophy, and dysfunction, possibly via inhibition of the TGF-beta/Smad3 signaling pathway.

A characteristic peculiarity of the trigeminal sensory system is the presence of two distinct populations of primary afferent neurons. Most of their cell bodies are located in the trigeminal ganglion (TG) but part of them lie in the mesencephalic trigeminal nucleus (MTN). This review compares the neurochemical content of central versus peripheral trigeminal primary afferent neurons. In the TG, two subpopulations of primary sensory neurons, containing immunoreactive (IR) material, are identified: a number of glutamate (Glu)-, substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)-, cholecystokinin (CCK)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and galanin (GAL)-IR ganglion cells with small and medium-sized somata, and relatively less numerous larger-sized neuropeptide Y (NPY)- and peptide 19 (PEP 19)-IR trigeminal neurons. In addition, many nitric oxide synthase (NOS)- and parvalbumin (PV)-IR cells of all sizes as well as fewer, mostly large, calbindin D-28k (CB)-containing neurons are seen. The majority of the large ganglion cells are surrounded by SP-, CGRP-, SOM-, CCK-, VIP-, NOS- and serotonin (SER)-IR perisomatic networks. In the MTN, the main subpopulation of large-sized neurons display Glu-immunoreactivity. Additionally, numerous large MTN neurons exhibit PV- and CB-immunostaining. On the other hand, certain small MTN neurons, most likely interneurons, are found to be GABAergic. Furthermore, NOS-containing neurons can be detected in the caudal and the mesencephalic-pontine junction portions of the nucleus. Conversely, no immunoreactivity to any of the examined neuropeptides is observed in the cell bodies of MTN neurons but these are encircled by peptidergic, catecholaminergic, serotonergic and nitrergic perineuronal arborizations in a basket-like manner. Such a discrepancy in the neurochemical features suggests that the differently fated embryonic migration, synaptogenesis, and peripheral and central target field innervation can possibly affect the individual neurochemical phenotypes of trigeminal primary afferent neurons.

A defective adenovirus (Ad) type 5 E1- vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd31 respectively. In human fibroblasts infected with RAd35 beta-galactosidase (beta-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with beta-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive beta-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV.

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.

Chinese hamster X mouse somatic cell hybrids segregating mouse chromosomes were examined for their mouse chromosome content using trypsin-Giemsa (GTG) banding and Hoechst 33258 staining techniques. Simultaneously, they were scored for the presence of 24 mouse enzymes. The results confirm the assignments of 11 genes previously mapped by sexual genetics: Dip-1 and Id-1 to chromosome 1; Pgm-2 and Pgd to 4; Pgm-1 to 5; Gpi-1 to 7; Gr-1 to 8; Mpi-1 and Mod-1 to 9; Np-1 and Es-10 to 14. They also confirm chromosomally the assignments of 3 genes that were made by other somatic cell genetic studies: Aprt to 8; Hprt and alpha-gal to the X chromosome. But most importantly, four enzyme loci are assigned to four chromosomes that until now were not known to carry a biochemical marker which is expressed in cultured cells: Trip-1 to 10; Dip-2 to 18; Acp-1 to 12; and Ak-1 to 2. Cytogenetic examination of clones showing discordant segregation of HPRT and A-GAL, suggested the assignment of alpha-gal to region XE leads to XF of the mouse X chromosome. The cytologic studies provide a comparison between data from sexual genetics and somatic cell hybrids and validate hybrid cell techniques. They provide evidence of the reliability of scoring chromosomes by GTG and Hoechst staining and stress the importance of identifying clones with multiple chromosome rearrangements. Striking examples of norandom segregation of mouse chromosomes were observed in these hybrids with preferential retention of 15 and segregation of 11 and the Y chromosome.

Flagellar mutants in Escherichia coli were obtained by selection for resistance to the flagellotropic phage chi. F elements covering various regions of the E. coli genome were then constructed, and, on the basis of the ability of these elements to restore flagellar function, the mutations were assigned to three regions of the E. coli chromosome. Region I is between trp and gal; region II is between uvrC and aroD; and region III is between his and uvrC. F elements carrying flagellar mutations were constructed. Stable merodiploid strains with a flagellar defect on the exogenote and another on the endogenote were then prepared. These merodiploids yielded information on the complementation behavior of mutations in a given region. Region III was shown to include at least six cistrons, A, B, C, D, E, and F. Region II was shown to include at least four cistrons, G, H, I, and J. Examination of the phenotypes of the mutants revealed that those with lesions in cistron E of region III produce "polyhooks" and lesions in cistron F of region III result in loss of ability to produce flagellin. Mutants with lesions in cistron J of region II were entirely paralyzed (mot) mutants. Genetic analysis of flagellar mutations in region III suggested that the mutations located in cistrons A, B, C, and E are closely linked and mutations in cistrons D and F are closely linked.

The GALGALGALGALGAL cluster genes in the host chromosome partially escaped the normal control; a yeast that harbors the plasmid bearing the GALGAL cluster genes at a low but significant level in the absence of galactose. If the GALGALGal-1-P uridylyl transferase encoded by GALGAL enzymes was not detected if GALGALGALGAL enzymes observed in the GALGALGALGAL cluster genes was also isolated, and when amplified together with GALGAL enzymes was observed, suggesting that the balanced synthesis of two regulatory proteins was essential to maintain the repressed state of the GAL cluster genes.