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Biomedical subjects

N Hopkins

Publications and source records attributed to N Hopkins.

At least 19 recordsLinked to original sources
PubMed1997

Expression of zebrafish rag genes during early development identifies the thymus.

Recent experiments have demonstrated that zebrafish is a vertebrate in which it is possible to carry out large-scale mutagenic screens to identify genes involved in specific developmental pathways. To follow development of the immune system in zebrafish, we have analyzed the expression of the recombination activating genes, rag1 and rag2, which we have previously isolated and characterized. These genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate markers to follow the development of organs containing these cells. By whole-mount in situ hybridization, we detected expression of both rag genes in a paired organ in the head, beginning on the fourth day after fertilization. Histological examination of this organ indicated that it corresponds to the thymus, as described for other fish, an organ that has not previously been identified in zebrafish. By histological analysis, the thymus primordium appears at 54 hr but does not enlarge significantly until 30 hr later. The thymus continues to enlarge and reaches its mature histological organization at 1 month. The pronephros, the major hematopoietic organ in the adult fish, begins to develop hematopoietic tissue about 2 weeks after fertilization. By 1 month, mature lymphocytes are distinguishable in the tissue surrounding renal tubules. Lymphocytes appear in the kidney too late for screening by whole-mount in situ hybridization; however, the pattern of rag1 expression in the thymus forms the basis of an assay for mutations affecting development of the thymus or its constituent lymphocytes.

Animals↗
PubMed1997

Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.

Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.

Amino Acid Sequence↗
PubMed1997

Delayed in vitro fertilization of zebrafish eggs in Hank's saline containing bovine serum albumin.

In zebrafish it is possible to create viable diploid fish whose genomic DNA is derived only from the female parent (parthenogenesis) or, as was more recently shown, only from the male (androgenesis). Androgenesis requires holding zebrafish eggs in an inactivated state in vitro for an hour or more. Previously this was achieved by placing the zebrafish eggs in ovary fluid obtained from rainbow trout (Onchorhynchus mykiss) or coho salmon (Onchorhynchus kisutch). Here we report that adding bovine serum albumin (BSA) to Hank's buffered saline prevents zebrafish egg activation in vitro. Of the zebrafish eggs placed in Hank's saline plus 0.5% BSA, 85% +/- 8.7% were fertilizable after incubation for one hour at room temperature (23 degrees C). Longer incubations are possible but with lower efficiency of fertilization. This technique not only could facilitate androgenesis, but also might be useful when making transgenics by microinjection, when performing antibody or RNA injections before fertilization, or for studying the mechanisms of egg activation in zebrafish.

Animals↗
PubMed1996

Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development.

Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.

Amino Acid Sequence↗
PubMed1996

Insertional mutagenesis and rapid cloning of essential genes in zebrafish.

Large-scale chemical mutagenesis screens in zebrafish have led to the isolation of thousands of lethal mutations in genes that are essential for embryonic development. However, the cloning of these mutated genes is difficult at present as it requires positional cloning methods. In Drosophila, chemical mutagenesis screens were complemented with P-element insertional mutagenesis which facilitated the cloning of many genes that had been identified by chemical lesions. To facilitate the cloning of vertebrate genes that are important during embryogenesis, we have developed an insertional mutagenesis strategy in zebrafish using a retroviral vector. Here, in a pilot screen of 217 proviral insertions, we obtained three insertional mutants with embryonic lethal phenotypes, and identified two of the disrupted genes. One of these, no arches, is essential for normal pharyngeal arch development, and is homologous to the recently characterized Drosophila zinc-finger gene, clipper, which encodes a novel type of ribonuclease. As it is easy to generate tens to hundreds of thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting vertebrate developmental and cellular processes.

Amino Acid Sequence↗
PubMed1996

Highly efficient germ-line transmission of proviral insertions in zebrafish.

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

3T3 Cells↗
PubMed1996

Requirements for green fluorescent protein detection in transgenic zebrafish embryos.

We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 alpha enhancer/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and non-fluorescent lines, in order to determine how this affects the production of detectable levels of GFP in the zebrafish embryo. Additionally, we have injected purified recombinant GFP into embryos to determine the intracellular GFP concentration required for detection, both when all of the cells in the embryo contain GFP and when only a few do.

Animals↗
PubMed1996

Seeking influence through characterizing self-categories: an analysis of anti-abortionist rhetoric.

This paper presents an analysis of an anti-abortionist's speech to a medical audience. It is shown that central to the speech is the way in which the speaker defines the context of the abortion debate and hence the categories of people involved. In particular, the speaker construes himself as a member of a common in-group with his audience, construes the entire audience as part of an anti-abortion category and claims that abortion is in contradiction with the defining features of the audience's medical identity on a series of levels. This analysis is used to make two suggestions. Firstly, following self-categorization theory, that the ways in which self-categories are defined may be central to the process of mass social influence. Secondly, however, self-categories may not be specified by intra-psychic processes but rather are discursively constructed and argued over. The implications of such a position for future research on self-categorization and category salience are discussed.

Abortion Applicants↗
PubMed1996

Transcriptional specificity of the pluripotent embryonic stem cell.

The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding sequences for a beta-galactosidase-neomycin phosphotransferase hybrid protein. Integrated proviruses, which disrupted expressed cellular genes, were selected in the presence of G418. ES clones expressing regulated beta geo fusion genes were identified by changes in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining after in vitro differentiation. Thirty-one of 191 clones tested (16%) exhibited regulated expression of beta geo protein. Seven genes disrupted by U3 beta geo were passed into the germline, and expression of the beta geo fusion genes was analyzed in vivo, including inserts disrupting the Eck and REX-1 genes. In each case, genes trapped in cultured ES cells were expressed in the inner cell mass of preimplantation embryos, and changes in lacZ expression during in vitro differentiation were also observed during early development. Thus, cultured ES cells maintain, to a considerable extent, the transcriptional specificity of the pluripotent cells of the preimplantation embryo. As a consequence, in vitro screens utilizing gene traps provide a rapid and accurate means to identify and disrupt developmentally regulated genes.

Animals↗
PubMed1995

Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S.

Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD+ by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD+. These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalyst, His444'. A pKa of 5.1 was estimated for Cys44 in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S. Ferricyanide stoichiometrically reoxidizes the FADH2 of both altered forms of the enzyme.

Cysteine↗
PubMed1995

Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides.

The cysteines that comprise the active site disulfide in lipoamide dehydrogenase have been individually mutated to a serine residue to give the altered enzymes, C44S and C49S, making it possible to study the redox behavior of the FAD in the absence of the disulfide. The redox potential of the FAD in C44S and C49S was -379 and -345 mV, respectively, at pH 7.0, 25 degrees C. A plot of the redox potential as a function of pH for C49S gave slopes of 57 mV/pH from pH 5.0 to 7.9 and 10 mV/pH from pH 7.9 to 8.8. The plot of the redox potential as a function of pH for C44S gave slopes of 70 mV/pH from pH 5.0 to 7.9 and 4 mV/pH from pH 7.9 to 8.38. The change in the slope at pH 7.9 is associated with the ionization (pKa) of the FADH2 to FADH- in the reduced form of both enzymes. These determinations show that the redox potential of the FAD in C49S, in C44S, and in wild type enzyme is modulated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide, and that the flavin is bound more tightly to the oxidized forms of these enzymes than to the reduced forms. The redox potentials of these enzymes determined using NADH and NADPH at pH 7.6, 25 degrees C are as follows: C44S, -350 mV, -369 mV; C49S, -328 mV, -353 mV, respectively. Thus, pyridine nucleotide binding raises the redox potential of the flavin, showing that both substrates bind more tightly to the reduced form of the enzymes, as well as tighter binding of NADH to the enzymes than that of NADPH. Kd values for the binding of NADH and NADPH to oxidized C44S and C49S were determined in pre-steady-state kinetics at pH 7.6 and 25 degrees C, which were monophasic when NADPH was the reductant and biphasic with NADH. The binding constants for NADPH were 660 microM for C44S and 500 microM for C49S; using NADH, the binding constants were 137 microM for C44S and 23 microM for C49S. Fluorescence and absorbance spectrophotometry were used to determine the binding of NAD+ to the oxidized forms of the enzymes as 275 microM and 270 microM for C44S and C49S, respectively.

Binding Sites↗
PubMed1995

The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos.

The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GFP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition. Fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. Fluorescence is most visible between 24-36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos.

Animals↗
PubMed1994

Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish.

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

Animals↗
PubMed1994

lacZ expression in germline transgenic zebrafish can be detected in living embryos.

Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish. We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element. Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos. The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons. Expression patterns were stable through the F2 generation in the three lines studied to date. The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG). Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised. These observations should prove useful for a variety of studies in zebrafish.

Animals↗
PubMed1994

HIV-1 infection at two public psychiatric hospitals in New York City.

Seroprevalence for HIV-1 was anonymously evaluated between November 1989 and July 1991 among severely mentally ill patients at two public psychiatric hospitals in New York City. The study population consisted of new admissions and long-stay patients aged 18-59. Of 1116 eligible patients, usable samples were obtained from routine blood drawings on 971 (87%). Seroprevalence was comparable among men (5.2%) and women (5.3%). Age did not predict seropositivity. Men with a recorded history of homosexual behaviour or injection drug use were, respectively, 1.8 and 2.0 times more likely to be seropositive than men without these histories. Women with a recorded history of injection drug use were 4.0 times more likely to be seropositive than women without such a history. Ethnicity was not predictive for men, but Black women were 2.4 times more likely to be HIV-1 positive than non-Black women. Severely mentally ill inpatients had a substantial rate of HIV-1 seropositivity, indicating a need for additional testing, education and counselling efforts for this population.

Adolescent↗