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Gene targeting in mouse embryonic stem cells has become the 'gold standard' for determining gene function in mammals. Since its inception, this technology has revolutionized the study of mammalian biology and human medicine. Here I provide a personal account of the work that led to the generation of gene targeting which now lies at the centre of functional genomic analysis.
The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.
Functional as well as structural assessment of the basal forebrain has mostly focused on the dorsal caudate and putamen in axial slices where they are easily outlined or their centers located with stereotaxic methods. The more ventral extent of the basal forebrain, where the irregular form and indistinct boundaries of the nucleus accumbens and substantia innominata are difficult to trace and where the brain's ventral surface may contribute partial volume artifacts to measurement, has been less studied. We present a method based on coronal sections, landmarks placed on clearly visible anchor points, and the computational technique of thin-plate spline warping which allows the alignment of groups of individuals to common coordinates for pixel-by-pixel statistical mapping. The reliability of the landmarks across independent raters yields a median absolute difference of 1.3-1.6 mm. The validity of the method is confirmed by variance maps which reveal significant decreases in variance over spindle and bounding box alignment.
This study reports the expression and functional characterization of the rat analogue of the human complement regulatory molecule CD59. We here describe the expression in chinese hamster ovary (CHO) cells of rat CD59 and a modified rat CD59 in which an N-glycosylation site at Asn-16 has been deleted by point mutation. The complement-inhibiting capacity of these two forms of rat CD59 has been analysed and compared. Expressed rat CD59 efficiently inhibited complement lysis of CHO cells when rat serum was used as a source of complement and also inhibited lysis by complement from all other species tested, confirming that rat CD59 displayed little or no species restriction of activity. Blocking of expressed rat CD59 with a monoclonal antibody abrogated the inhibition of lysis for all sources of complement, confirming that the expressed molecule was responsible for the protection. The glycosylation mutant had a much reduced molecular weight on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12,000 MW as compared with 20,000-28,000 MW for unmutated), confirming that it was unglycosylated. However, the glycosylation mutant had complement-inhibitory activity which was at least as potent as that of the unmutated molecule, demonstrating that the large, N-linked carbohydrate moiety was not required for function.
We have demonstrated that tumor-infiltrating T cells play a crucial role in tumor growth. In order to investigate the precise function of tumor-infiltrating cells, we avoided the use of any enzyme for separation of tumor-infiltrating cells from the solid tumor mass and established a new system in which we could easily obtain functionally active tumor-infiltrating cells in large amounts from the peritoneal cavity of T-9 gliosarcoma-sensitized rats. In this system, viable T-9 cells were intraperitoneally administered and peritoneal exudate cells were recovered. Importantly, cells obtained from the intraperitoneal cavity are phenotypically and functionally indistinguishable from those obtained from subcutaneous tumor masses. Furthermore, injection of purified lymphocyte migration factor-b resulted in the accumulation of CD8 (+) cytotoxic T cells in the peritoneal cavity. Therefore, this system also provides a means by which the in vivo function of chemotactic factor can be tested.
Phenotypic and functional aspects of cellular cytotoxicity were investigated in patients after splenectomy. While assays testing for natural killer cell (NK) activity and antibody-dependent cellular cytotoxicity showed a significantly reduced (p less than 0.005 and p less than 0.025, respectively) activity of the relevant cell populations, lectin-dependent cellular cytotoxicity was either normal or, in one dilution of phytohemagglutinin, even significantly increased (p less than 0.025), as compared to healthy control persons. In a search for a possible explanation for this phenomenon, it was found that the total lymphocyte count was significantly increased (p less than 0.01) in patients after splenectomy, when compared with healthy control individuals. However, neither the absolute cell count nor the percentage of Leu7+ lymphocytes showed significant differences between splenectomized patients and controls. In contrast, the percentages of (Leu7+Leu2)+, OKT11+, OKT4+ and Leu2a+ lymphocytes were significantly decreased (p less than 0.0025, p less than 0.01 and p less than 0.005, respectively) in splenectomized patients with not only the percentage, but also the absolute number of DR+ lymphocytes being significantly increased (p less than 0.005 and p less than 0.05, respectively). We thus conclude that in patients after splenectomy NK activity is decreased which may possibly be due to the lack of a NK subset. In contrast, the overall percentages of T cells (OKT11+), T helper/inducer (OKT4+) and T suppressor cells (Leu2a+) were significantly decreased in patients after splenectomy, although this defect could be compensated by a significant increase in the absolute lymphocyte number.
Oestrogens have a major impact on reproductive function in both males and females. Two oestrogen receptor genes known as ERalpha (ESR1NR3A1) and ERbeta (ESR2NR3A2) have been cloned. Splice variant isoforms of the ERbeta gene have been identified in human, bovine and rodents and it has been suggested that the existence of these forms can influence oestrogen responsiveness. In the human, splicing of an alternative eighth exon results in the formation of a C-terminal variant called hERbetacx, or hERbeta2, but this isoform has not been identified in other species. The aim of the present study was to clone ERbeta cDNAs from primates so as to determine how closely they resembled the ERbeta isoforms found in the human. The two species studied were the stump-tailed macaque (Macaca arctoides), an Old World primate, and the common marmoset (Callithrix jacchus jacchus), a New World primate. Full length ERbeta (wild type, ERbeta1) cDNAs were cloned from macaque and marmoset; they encoded proteins of similar size to those found in human (59 and 54 kDa, long and short forms respectively) and shared significant sequence homology (97.5% in macaque and 93.8% in marmoset) with the human peptide sequence. Full length cDNAs homologous to the hERbeta2 variant were identified in both primates. Marmoset ERbeta2 was slightly shorter than that of human ERbeta2 (54 kDa compared with 55 kDa) and did not contain the peptide sequence used to raise an anti-hERbeta2 antibody. All the macaque ERbeta2 cDNAs contained 56 bp of intronic sequence which included an in-frame stop codon resulting in translation of a truncated protein ( approximately 35 kDa). In all three species, truncated, alternatively spliced mRNAs lacking exon 5 were isolated on multiple occasions from all tissue extracts. In transient transfection assays, ERbeta2-containing constructs were unable to induce transcription of an oestrogen response element (ERE) reporter plasmid in the presence of oestradiol. ERbeta1 from human, macaque and marmoset exhibited minor differences in their ability to induce transcription of the ERE reporter when incubated with different ligands (oestradiol, PPT, DPN, 5-alpha-androstane-3-beta, 17beta-diol (3betaAdiol), genistein) and this may be due to amino acid substitutions within their ligand binding domains. In conclusion, we have identified and cloned wild type ERbeta (ERbeta1) from macaque and marmoset and demonstrated that splice variant mRNAs homologous to hERbeta2 are formed in both species. The marmoset monkey, therefore, provides a suitable animal model in which to investigate the impact of ERbeta variant expression on tissue responsiveness to oestrogens.
In order to evaluate the functional aspect of myocardial neovascularization after grafting a free skeletal muscle flap onto the heart in the dog with an arterial anastomosis to the internal mammary artery (IMA), a gradual occlusion of the left anterior descending coronary (LAD) was induced. This was achieved implanting an Ameroidconstrictor on the LAD, that due to its hygroscopical material induces a gradual coronary occlusion. The occlusion of the LAD was angiographically confirmed in vivo. In one dog the selective angiographical demonstration of the IMA anastomosed to the graft was achieved. This showed retrograde filling of the LAD occluded by the Ameroidconstrictor. These findings were confirmed in all dogs by a microvascular corrosion cast preparation of the vasculature induced from the graft, that was differentiated from the coronaries by a different coloured casting material. The occluded LAD was filled in all preparations by the casting material applied into the IMA. Thus, grafting of a free skeletal muscle flap anastomosed to the IMA onto the dog's heart produces a functional myocardial neovascularization.
We have shown that Leishmania lipophosphoglycan (LPG) inhibits IL-1 beta gene expression in human monocytes. Here, we show that LPG can bind in a time-dependent manner and suppress endothelial cell activation, possibly via specific LPG domains. Endotoxin (10 ng/ml, 4 h) consistently caused endothelium to increase monocyte adhesion (approximately 20-fold). LPG pretreatment (2 microM, 2 h) completely blocked endotoxin-mediated monocyte adhesion. LPG did not grossly suppress endothelial functions because TNF-alpha- and IL-1 beta-mediated adhesion toward monocytes were not affected. Using four highly purified LPG fragments (namely, repeating phosphodisaccharide (PGM), phosphoglycan, phosphosaccharide core-lyso-alkyl-phosphatidylinositol (core-PI), and lyso-alkyl-phosphatidylinositol (lyso-PI)), we examined whether these fragments can independently inhibit endothelial adhesion. In contrast to that of intact LPG, neither the four LPG fragments (2 microM, 2 h) independently nor the co-addition of phosphoglycan and core-P1 fragments blocked the endotoxin-mediated adhesion to monocytes. To determine whether the fragments can reverse the effect of intact LPG, endothelial cells were first pretreated with the LPG fragments (10 microM, 15 min), followed by the addition of LPG (2 microM). All four LPG fragments fully reversed the effect of LPG. Simultaneous addition of LPG fragments and intact LPG caused only partial suppression (approximately 45%), while the addition of LPG fragments 14 min later had no reversal effect. Flow cytometry revealed that only core-P1 and lyso-P1 competitively inhibited (approximately 30%) LPG binding. Conversely, LPG competed with the binding of [3H]lyso-P1 (approximately 30%). Furthermore, mAb against the PGM reversed (approximately 70%) the effect of LPG. Thus, the lyso-P1 domain on LPG mediates binding to endothelial cells, whereas the PGM domain mediates the cell inhibitory effect.
The effect of finite trajectory length on single molecule rotational correlation functions has been studied by utilizing time series analysis and numerical simulations. Correlation functions obtained from the trajectories of length less than 100 times the correlation time constant (tau([script-l])) exhibit significant deviations from the true correlation function. The distributions of sample time constants (tau(F)) and stretching exponents (Beta(F)) are mapped by fitting a large number of rotational trajectories to stretched exponentials. As the trajectory length gets smaller, the distributions become broader and asymmetric and their mean values deviate from the true value predicted by pure rotational diffusion. Analysis based on higher order spherical harmonics is suggested as a method for minimizing the effect of the trajectory length. The distributions of time constants for different higher order spherical harmonics are also compared. While the focus of the paper is on rotational correlation functions, the general conclusions apply to any dynamical process that yields an exponentially decaying correlation function.
To further understand the evolutionary dynamics of the regulatory interactions underlying development, we expand on our previous analysis of hunchback and compare the structure and function of the tailless enhancer between Musca domestica and Drosophila melanogaster. Our analysis shows that although the expression patterns and functional protein domains of tll are conserved between Musca and Drosophila, the enhancer sequences are unalignable. Upon closer investigation, we find that these highly diverged enhancer sequences encode the same regulatory information necessary for Bicoid, Dorsal, and the terminal system to drive tll expression. The binding sites for these transcription factors differ in the sequence, number, spacing, and position between the Drosophila and Musca tll enhancers, and we were unable to establish homology between binding sites from each species. This implies that the Musca and Drosophila Bcd-binding sites have evolved de novo in the 100 million years since these species diverged. However, in transgenic Drosophila embryos the Musca tll enhancer is able to drive the same expression pattern as endogenous Drosophila tll. Therefore, during the rapid evolution of enhancer sequences individual binding sites are continually lost and gained, but the transcriptional output is maintained by compensatory mutations in cis and in trans.
The GATA family of zinc finger proteins are transcriptional regulators with critical functions in lineage differentiation and embryonic development. Based on structural and expression pattern comparisons, the GATA proteins have been subdivided into two groups. The first subgroup consists of GATA-1, -2, and -3, which are all highly expressed in the hematopoietic system, whereas GATA-4, -5, and -6 are present essentially in the heart and gut. We have isolated and functionally characterized the rat GATA-5 cDNA, which encodes a 45-kDa protein with 71%, 73%, and 97% homology to its amphibian, avian, and murine homologs, respectively. Northern blot analysis showed that rat GATA-5 is expressed in a dynamic pattern during embryonic and postnatal development. In the midgestation embryo, GATA-5 transcripts are most abundant in the heart and decrease dramatically in the postnatal heart; in contrast, GATA-5 expression is upregulated in the lung and gut during postnatal development. Functional studies with recombinant GATA-4, -5, and -6 proteins show that GATA-5 has preferential affinity for a subset of GATA elements found on cardiac promoters and differentially activate cardiac gene transcription. Structure-function analysis revealed the presence of an activation domain within the carboxy terminal region of GATA-5 that is essential for transcriptional regulation of target promoters. Linkage analysis localized Gata5 to distal mouse Chromosome (Chr) 2 in a conserved linkage group with genes localized to rat Chr 3q43 and human Chr 20q13.2-q13.3. The results suggest that GATA-5 may have specific downstream targets and that GATA-4, -5, and -6 can only partially substitute for each other in cardiogenesis. Thus, Gata5 probably plays a specialized evolutionary conserved role in cardiac development.
Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.
Thymic cell populations from 12 patients displaying myasthenia gravis were submitted to a phenotypic and functional study. Immunofluorescence analysis of thymic sections revealed the presence in germinal centers of B lymphocytes expressing the B cell markers--CD19, CD21, IgD, or IgM. After T cell and macrophage depletion of thymic single cell suspensions, B cell-enriched populations were isolated. Enriched B cells expressed at variable levels activation markers such as CD71, 4F2, CD23, and B8.7, indicating that a marked proportion of them are activated. Moreover, addition of B cell growth factor 12kDa and to a lesser extent of rIL-2 induced a spontaneous proliferation of these B cell populations. These functional and phenotypic signs of activation may reveal the first steps of an autoimmune response against acetylcholine receptor as enriched B cell populations have the capacity to spontaneously secrete anti-acetylcholine receptor antibody.
The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.
Long Evans cinnamon (LEC) rat is an animal model for human Wilson disease (WD) due to a deletion in Atp7b, the copper transporter defective in WD patients. Previously, we have demonstrated presence of an alternative product termed PIneal Night-specific ATPase (PINA) generated by an intronic promoter in Atp7b gene. Analysis of LEC rat in this study demonstrates that PINA is absent in the LEC pineal establishing its usefulness for investigating PINA function. Studies of the LEC pineal, however, revealed an additional defect in serotonin N-acetyltransferase (NAT), the key enzyme in melatonin production. Linkage studies confirm that the NAT phenotype is entirely independent of PINA mutation in the pineal gland of LEC rats, and sequence analysis demonstrates that NAT defect is due to a point mutation in NAT coding region. In addition, we demonstrate that the cinnamon coat color of the LEC rat is unlinked to PINA and NAT deficiencies in these animals. To facilitate further functional analysis of PINA in pineal physiology, we crossed LEC rats with PVG rats that are wildtype for PINA, NAT and coat color, and obtained rats that are defective only in PINA/Atp7b locus (termed LPP rats) and normal for NAT activity and coat color. Furthermore, we have identified the deletion breakpoint of Atp7b gene in LPP rats, which allows simplified genotyping of mutant animals. The separation of PINA mutation from both NAT and coat color mutations in the new LPP rats will permit better functional studies of PINA in pineal circadian physiology.
The p65 subunit of the NF-kappa B transcription factor contains in its C-terminal 120 amino acids at least two transcription activation domains. One domain (TA1) is contained within only the 30 C-terminal amino acids. Structural studies employing CD and NMR spectroscopy revealed that the TA1 domain is unstructured. NMR analysis of a protein corresponding to the C-terminal 123 amino acids also showed a random coil conformation. However, a portion of TA1 was found to adopt an alpha-helical conformation in the presence of hydrophobic solvents. Transcriptional analysis of point mutants revealed the functional importance of two evolutionary conserved sequence repeats, which are located in the conditionally alpha-helical region of TA1. These repeats acted synergistically in transcription activation. The inhibitory effect of some mutants indicated secondary structure constraints on TA1 in intact cells. Inverting the sequence of two acidic activation domains significantly reduced their transactivating potential, suggesting that amino acid composition is not solely essential for activity; a defined primary structure is necessary as well. Acidic sequence motifs related in primary structure and squelching activity to those of TA1 are present in the activation domains of VP16, c-Rel, and several other transcription factors. We propose a model suggesting that primarily unstructured acidic activation domains can adopt a secondary structure upon contacting their target molecules by an "induced fit" mechanism.
We investigated the phenotype and function of thyroid tumor-, metastatic lymph node-, and peripheral blood-derived T lymphocytes of four patients with papillary thyroid cancer. Both phenotypic analysis of freshly isolated cells and clonal analysis, using a high efficiency cloning technique, were performed. For comparison, intrathyroid and peripheral blood T lymphocytes derived from patients with autoimmune thyroid diseases (Graves' disease and Hashimoto's thyroiditis) have been studied. In papillary cancer, the phenotype of thyroid and lymph node-derived T lymphocytes did not differ from that of peripheral blood lymphocytes of the same patients or lymphocytes from normal peripheral blood. At variance with respect to autoimmune thyroid disease, activation markers were poorly represented. The functional analysis of T cell clones showed similar proportions of interleukin-2-producing (helper-inducer) clones in thyroid, lymph node, and peripheral blood, slightly lower than in Hashimoto's thyroiditis and slightly higher than in Graves' disease. With regard to effector function, we found lower proportion of clones with cytolytic activity in a lectin-dependent assay compared to that in Hashimoto's thyroiditis. Interestingly, however, the proportions of cytolytic clones displaying cytolytic activity against the neoplastic cell line K562 (natural killer-like activity) or fresh unrelated tumor cells (lymphokine-activated killer activity) were relatively high in thyroid cancer infiltrates.