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An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.
The projection from the superficial layers of the tectum to the pretectal complex in the cat was examined using the retrograde transport of wheat germ agglutinin-horseradish peroxidase. Restricted injections were made into different parts of the pretectum. Neurons in the superior colliculus were found to be arranged in a mediolateral array that corresponds to the rostromedial to caudolateral array of their axon projections to the nucleus of the optic tract and posterior pretectal nucleus. These results suggest that similar parts of the retinotopic maps present in the pretectum and superior colliculus are connected. The labeled cells in the superior colliculus were located within the deep part of stratum griseum superficiale and the superficial part of stratum opticum, and were composed of multipolar cells, vertical fusiform cells and horizontal cells. We conclude from this study that the cells of origin in the superior colliculus originate in the same region to which the contralateral retinal Y-cells project and that have also the morphological diversity, as do other tecto-thalamic neurons.
The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus. Retroviruses and, especially, endogenous retroviruses are traditionally of the most developed models of viral carcinogenesis. Many endogenous retroviruses are implicated in neoplastic transformation of the cell. For instance, endogenous mouse mammary tumor virus of some inbred lines appears to be the only causative agent in these mammary cancers. Other even nonpathogenic murine endogenous retroviruses are involved in the origination of MCF-type recombinant acute leukosis viruses. Some endogenous retroviruses are implicated in the transduction or activation of cellular protooncogenes. Our interest in endogenous viruses is based on their ability to make cells resistant to exogenous retroviruses. Expression of their major envelope glycoprotein leads to cellular surface receptor blockage and imparts immunity to infection by the related leukemia retroviruses. This problem is quite elaborated for chicken endogenous virus RAV-O (7-9).(ABSTRACT TRUNCATED AT 250 WORDS)
Autoradiography of restriction digests of DNA labeled in early S phase indicates that replication of the amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells initiates within a 6.1-kilobase pair (kb) EcoRI-doublet located on the 3' side of the DHFR gene. To localize the DHFR origin fragment, synchronized CHOC 400 cells were either pulse labeled with [3H]thymidine in vivo or permeabilized and incubated with [32P]dATP under conditions that support limited chromosomal DNA replication. The temporal order of replication of amplified fragments was determined by hybridization of the in vivo or in vitro replication products to cloned fragments spanning the earliest-replicating portion of the DHFR domain. At the G1/S boundary, the labeled products derived from the replication of amplified sequences, either in whole or permeabilized cells, are distributed about an amplified 4.3-kb Xba I fragment that maps 14 kb downstream from the DHFR gene. As cells progress through the S phase, bidirectional replication away from this site is observed. These studies indicate that the 4.3-kb Xba I fragment contains the origin of replication associated with the amplified DHFR domain.
A new pathway for aerobic metabolism of 2-aminobenzoate which proceeds via anthranoyl-CoA has recently been revealed in a Pseudomonas strain KB740. This bacterial strain was found to contain a small 8.1-kbp plasmid pKB740 which appears to harbour the genes encoding for two key enzymes catalyzing the initial reactions of the pathway, 2-aminobenzoate coenzyme A ligase and 2-aminobenzoyl-coenzyme A monooxygenase/reductase. The evidence is as follows: The plasmid content of the culture varied by a factor of ten depending on the growth substrates; it was highest when cells were grown aerobically on 2-aminobenzoate. The plasmid pKB740 could be introduced into Escherichia coli strain JM83 by transformation. Wild-type E. coli and E. coli JM83 are unable to metabolize 2-aminobenzoate whereas the transformed E. coli JM83 cells could grow with this aromatic compound as sole organic substrate and oxidize it completely to CO2. The plasmid recovered from E. coli had the same restriction map as the original plasmid, but was dimerized. The two key enzyme activities were demonstrated in the transformed E. coli in sufficiently high amounts to explain growth. They appear to be regulated on the transcription level by induction; they were formed only during aerobic growth in the presence of 2-aminobenzoate, as in the parent Pseudomonas. The N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase was similar to the consensus sequence of the FAD binding site of different flavoenzymes. The data also prove that the enzyme with two flavin functions is a alpha 2 homodimer. Southern blotting of digested chromosomal and plasmid DNA and hybridization against a labelled 15-base oligonucleotide derived from the N-terminal amino acid sequence of 2-aminobenzoyl-CoA monooxygenase/reductase revealed that the gene for this enzyme is coded on the plasmid rather than on the chromosome. The gene was localized on a 3.2-kbp restriction fragment. The formation of 2-aminobenzoyl-CoA monooxygenase/reductase protein in transformed E. coli was demonstrated by Western blotting of proteins of cell extracts separated by SDS/PAGE. The enzyme protein band, which was stained by a procedure based on antibodies against 2-aminobenzoyl-CoA monooxygenase/reductase, was demonstrated in transformed E. coli.
Construction of the shuttle cloning vectors for Escherichia coli-Brevibacterium flavum system is described. Expression of the Sp/Sm resistance determinant derived from the Corynebacterium plasmid pCG4 was registered in Escherichia coli cells. The genetic determinant for Sp/Sm resistance was shown to be located in a 2.2 kb PstI-SphI fragment by the deletion analysis mapping in Escherichia coli cells. Using Escherichia coli as a host we cloned the unique 0.8 kb EcoRI-EcoRI fragment of Brevibacterium flavum bacteriophage phi BSh6 in the plasmids with dual replication origins. Blocking of the shuttle vector transfer to Brevibacterium flavum by the insertion of bacteriophage phi BSh6 DNA was observed. The deletion of entire phage fragment or a specific part of it made it possible introduction of plasmids harboured by Escherichia coli cells into Brevibacterium flavum. A potential vector for homologous DNA cloning in Brevibacterium flavum was constructed.
The nuclear target of the X zone of the cerebellar cortex was identified in rats as clusters of neurons scattered at the interface between the nuclei medialis (NM) and interpositus (NI). In a previous study, we had outlined these target neurons and termed them "interstitial cell groups" (icg). In order to determine whether the icg should be considered as part of either the NM or the medial NI, we analyzed two efferent pathways from the icg: their nucleocortical and nucleoolivary projections. These were compared to their homologues from the NM and the NI. This analysis is based on mapping retrograde cell labeling and anterograde terminal labeling following microinjections of tracers in either the cerebellar cortex, the cerebellar nuclei, or the inferior olive. Nucleocortical projections originating from the icg are of the three types described previously: a "reciprocal" projection to the ipsilateral X zone, a "nonreciprocal" projection to the ipsilateral A zone, and a "symmetrical" projection to the contralateral X zone. These features can be considered as the summed characteristics of the nucleocortical projections from the NM and from the medial NI. Nucleoolivary projections from the icg target the lateral-rostral portion of the dorsal accessory olive as well as the centrocaudal part of the medial accessory olive. These pathways converge with the nucleoolivary projections from the medial NI and from the NM, respectively. The icg receives olivary afferents from both the regions of the dorsal and medial accessory olives to which it projects. On the basis of similarities shown here between the two types of efferents originating from the icg and those from the NM as well as the medial NI, the icg may be regarded as a "mosaic" of neuron clusters alternately belonging to the NM and the medial NI. Therefore, the icg would be reciprocally connected with the inferior olive.
Neural crest cells are derived from a population of multipotent stem cells within the neural tube. They emerge shortly after neural tube closure, migrate extensively in the embryo and localize in numerous sites, where they differentiate into neurons and glia of the peripheral nervous system, cartilage and bone of the face, melanocytes and various other cell types. This review summarizes recent experiments from our laboratory delineating the origin and lineage of avian neural crest cells. Neural crest cells arise from the ectoderm, which also gives rise to presumptive epidermal, placodal and neural tube cells. Fate mapping experiments have demonstrated that the neural crest arises at the juncture between presumptive epidermis and the neural plate. Inductive interactions between these two early tissues can generate neural crest cells, suggesting that signals travel through the epidermis to generate neural crest cells prior to neural tube closure. Injection of lineage tracer into individual cells reveals that a single neural fold can form all ectodermal derivatives (i.e., epidermis, neural tube, neural crest). Even after neural tube closure, neuroepithelial cells have the capacity to form multiple neural crest and neural tube derivatives, including both dorsal and ventral phenotypes, suggesting that neural tube and neural crest cells share a common precursor. Further evidence that neural crest and neural tube cells are intimately related comes from experiments in which the cranial neural folds are ablated. The remaining neural tube cells have the capacity to regulate, at least for a limited time, to compensate for missing neural crest cells. These experiments suggest that the early neuroepithelium has no clear segregation with respect to the neural tube or neural crest. With time, dorsalizing and ventralizing signals may cause neural tube cells to acquire specific cell fates.
Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE gamma) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE gamma was determined. The nature protein is composed of 318 amino acids with a calculated Mtau of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in the Vmax and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.
A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions.
Antigen-specific rat mouse T-cell hybridomas, prepared by fusing the BW5147 AKR thynoma with helper T cells from rats immunized with chicken ovalbumin, express both the rat and mouse major histocompatibility complex gene products. Using these functionally defined T-cell hybridomas, we have devised a technique based on the differential staining of the heterochromatin region of the rat and mouse chromosomes to distinguish the species of origin of the chromosomes. This technique should prove useful for screening potential hybrid cell lines and should aid in differentiating mouse from rat chromosomes for gene mapping studies.
A key feature of perception is that the interpretation of a single, continuously available stimulus can change from time to time. This aspect of perception is well illustrated by the use of ambiguous figures that can be seen in two different ways. When people view such a stimulus they almost universally describe what they are seeing as jumping between two states. If it is agreed that this perceptual phenomenon is causally linked to the activity of nerve cells, the state jumps would have to occur in conjunction with changes in neural activity somewhere in the nervous system. Our experiments suggest that hippocampal place cells are part of a perceptual system. We conducted variations of a 'cue-card rotation' experiment on rats in which the angular position of a prominent visual stimulus on the wall of cylinder is changed in the rat's presence. The three main results are that (i) place-cell firing fields remain stationary if the cue is rotated by 180 degrees, so the relation between the cue and the field is altered; (ii) firing fields rotate by 45 degrees when the cue is rotated by 45 degrees, so the relation between the field and the card is maintained; and (iii) if the cue is first rotated by 180 degrees and then rotated in a series of 45 degrees steps, the field winds up at a different angular position relative to the card when the card is back in its original position. Thus, place cells can fire in two different ways in response to a continuously viewed stimulus. We conclude that place cells reveal that the hippocampal mapping system also has properties expected of a perceptual system.
The tumor phenotype is associated with the rearrangement of genetic information and the altered expression of many gene products. In this review, genes associated with the tumor phenotype have been arranged on the human gene map and indicate the extent to which the tumor phenotype involves the human genome. Nonrandom chromosomal aberrations that are frequently observed in tumors are presented. Altered metabolic demands of the tumor cell are reflected in altered gene expressions of a wide range of enzymes and other proteins, and these changed enzyme patterns are described. The study of oncogenes increasingly suggests that they may be significant in certain cancers, and the assignment of these genes has been tabulated. The biochemical and metabolic changes observed in tumors are complex; studying the patterns and interactions of these changes will aid our genetic understanding of the origins and development of tumors.
Neurons of the Drosophila larval brain are formed by a stereotyped set of neuroblasts. As differentiation sets in, neuroblast lineages produce axon bundles that initially form a scaffold of unbranched fibers in the center of the brain primordium. Subsequently, axons elaborate interlaced axonal and dendritic arbors, which, together with sheath-like processes formed by glial cells, establish the neuropile compartments of the larval brain. By using markers that visualize differentiating axons and glial cells, we have analyzed the formation of neuropile compartments and their relationship to neuroblast lineages. Neurons of each lineage extend their axons as a cohesive tract ("primary axon bundle"). We generated a map of the primary axon bundles that visualizes the location of the primary lineages in the brain cortex where the axon bundles originate, the trajectory of the axon bundles into the neuropile, and the relationship of these bundles to the early-formed scaffold of neuropile pioneer tracts (Nassif et al. [1998] J. Comp. Neurol. 402:10-31). The map further shows the growth of neuropile compartments at specific locations around the pioneer tracts. Following the time course of glial development reveals that glial processes, which form prominent septa around compartments in the larval brain, appear very late in the embryonic neuropile, clearly after the compartments themselves have crystallized. This suggests that spatial information residing within neurons, rather than glial cells, specifies the location and initial shape of neuropile compartments.
A combined approach of cDNA cloning and direct oligonucleotide mapping of TGF-beta 1 mRNA from several human cell lines has revealed that the major human TGF-beta 1 transcript is 381 bases shorter than originally reported, and that the reduced mRNA size is due to polyadenylation from an ATTAAA signal at position 2136 rather than use of the expected AATAAA signal at position 2517. Moreover, there is no evidence for a significant amount of structural heterogeneity, as a result of alternative polyadenylation, in the human TGF-beta 1 transcripts. Considering that the 381-base domain is not part of the major human TGF-beta 1 mRNA, we analyzed this sequence for potential transcriptional regulatory elements. We have identified a 16-base pair domain which contains three putative phorbol ester responsive elements (TREs) based on homology to the TRE consensus sequence. We also show that this 16-base pair fragment confers phorbol ester responsiveness to the chloramphenicol acetyltransferase gene after transient transfection of the heterologous construct in NIH-3T3 cells. The identification of a TRE immediately downstream of the last TGF-beta 1 exon suggests that a 3' enhancer may play an important role in human TGF-beta 1 gene transcription, and suggests a basis for growth factor-mediated regulation of TGF-beta 1 expression by activation of protein kinase C.
We have used carbocyanine dyes to fate map the primitive streak in the early chick embryo, from stages 3+ (mid-primitive streak) to 9 (8 somites). We show that presumptive notochord, foregut and medial somite do not originate solely from Hensen's node, but also from the anterior primitive streak. At early stages (4- and 4), there is no correlation between specific anteroposterior levels of the primitive streak and the final position of their descendants in the notochord. We describe in detail the contribution of specific levels of the primitive streak to the medial and lateral halves of the somites. To understand how the descendants of labelled cells reach their destinations in different tissues, we have followed the movement of labelled cells during their emigration from the primitive streak in living embryos, and find that cells destined to different structures follow defined pathways of movement, even if they arise from similar positions in the streak. Somite and notochord precursors migrate anteriorly within the streak and pass through different portions of the node; this provides an explanation for the segregation of notochord and somite territories in the node.
DNA from the E. coli replicative origin binds with high affinity to outer membrane preparations. Specific binding regions are contained within a 463 bp stretch of origin DNA between positions -46 and +417 on the oriC map. This region of DNA contains an unusually high number of GATC sites, the recognition sequence for the E. coli DNA adenine methylase. We show here that oriC DNA binds to membrane only when it is hemimethylated. The E. coli chromosomal origin is hemimethylated for 8-10 min after initiation of replication, and origin DNA binds to membranes only during this time period. Based on these results, we propose a speculative model for chromosome segregation in E. coli.