Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat

We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda. A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping. Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR). The remaining seven clones are all different and contain various rearrangements. Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion. The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA. The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element. We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat. We have found that there are several "free" 412 LTRs in genomic DNA from D. melanogaster strains Canton S and Oregon R, and from D. melanogaster tissue culture cells. We have cloned and sequenced one of these free LTRs. It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long. This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion.     

Developmental studies of lethality associated with the antennapedia gene complex in Drosophila melanogaster

A number of dominant homoeotic mutations are localized to the proximal right arm of chromosome 3 of Drosophila melanogaster and are thought to represent members of a gene complex that controls normal determinative decisions in the head and thorax. We have designated this complex the Antennapedia gene complex (ANT-C). Developmental studies were done to investigate the nature of the lethality associated with members of two of the complementation groups within ANT-C. The first complementation group, represented by the mutant Multiple Sex Combs (Msc) is characterized by embryonic lethality when heterozygous with a deletion of the ANT-C. The second complementation group consists of Antennapedia (Antp), Antennapedia-Extra Sex Combs (Antp^Scx), and the lethals recovered as revertants of Antp^Ns. When heterozygous for a deletion of the ANT-C or in heterozygous condition with each other, the members of this group show effective lethal phases spanning from embryo-larval boundary to late larval stages. Wakimoto and Kaufman (1981) show that the Antp⁺ gene acts to establish normal determinative states in the thorax. In the present work, transplantation of eye-antennal disks from lethal individuals heterozygous for two different Antp^Ns revertant chromosomes into wild-type hosts allowed the assessment of the function of the Antp⁺ allele in the antenna. Since these transplants formed only antennal structures and showed no evidence of the antennal → leg transformation seen in Antp^Ns controls, we conclude that the wild-type function of the Antp locus is not necessary for the establishment and/or maintenance of the antennal determined state. We suggest that regulatory mechanisms associated with the Antp⁺ structural gene normally function both to allow its expression in the thorax and to repress it in the antenna.     

Defects in embryogenesis in mutants associated with the antennapedia gene complex of Drosophila melanogaster

Embryogenesis in individuals with mutations or deficiencies of the genes in the polytene interval 84A-84B1,2 of Drosophila melanogaster was examined using scanning electron microscopy (SEM). The developmental function of this region of chromosome 3 is of particular interest since it contains the Antennapedia Gene Complex (ANT-C), a gene cluster that includes the homoeotic proboscipedia (pb), Sex combs reduced (Scr), and Antennapedia (Antp) loci. The results of SEM studies, clonal analyses, and temperature-shift experiments show that the fushi tarazu (ftz) and zerknullt (zen) genes, which map between pb and Scr, are involved in processes initiated during embryogenesis. The activity of ftz+ appears to be required within the first 4 hr of development for the establishment of the proper number of segments in the embryonic germ band. Individuals with ftz mutations or deficiencies produce only half the normal number of segments. Each of the segments is twice the normal width and is apparently comprised of cells that would normally form two separate metameres. The zen allele is required from about 2-4 hr of embryogenesis. Mutations of this gene result in disturbances of morphogenetic movements during gastrulation. The mutant phenotype is characterized by the absence of the optic lobe, defects in involution of the head segments, and in some cases, failure of germ band elongation. A requirement during embryogenesis for the activities of other genes residing in the 84A-84B1,2 polytene interval is suggested by the phenotypes of individuals heterozygous or homozygous for chromosomal deficiencies. Using the deficiencies Df(3R)AntpNs+R17, Df(3R)Scr, and Df(3R)ScxW+RX2, we examined the effects of deleting the distal portions or all of the 84A-84B1,2 interval. The defects in deletion heterozygotes suggest that the wild-type activity of some gene(s) other than zen, within or just adjacent to the 84B1,2 doublet, is required to complete normal head involution. The deletion of all the loci in the 84A5-84B1,2 interval results in grossly abnormal morphology and morphogenesis of the gnathocephalic appendages of the embryo. From these studies we conclude that mutations and deficiencies of genes associated with the ANT-C have profound effects on embryogenesis. The mutant phenotypes suggest, in addition to ensuring proper segment identity, the wild-type alleles of the 84A-84B1,2 genes are necessary for normal segmentation and elongation of the germ band and normal head involution.     

Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster.

Tritiated thymidine was administered at various times, and for various lengths of time, during the larval stages of Drosophila melanogaster. The thymidine was incorporated into DNA and was subsequently detected by autoradiography. These procedures allowed identification of those cells undergoing DNA replication at a particular time and also allowed determination of subsequent changes in relative position of these cells and some of their progeny. An analysis of these data has elucidated characteristic patterns of cell division and cell movement in the formation of the adult nervous system during postembryonic developmental stages.     

Developmental compartments in the Drosophila melanogaster wing disc

Distribution of the enzyme aldehyde oxidase (AO) within the pouch of the mature wing disc is precise and differential. General locations of compartmental boundaries have been identified by fate mapping and studies of AO distribution. The suspected locations of the boundaries were verified by analyzing the distribution of AO-negative cells within an AO-stained background in gynandromorphs and in X-ray-induced clones of AO-negative cells. The anterior/posterior border appeared slightly anterior to the junction of the AO⁺ anterior presumptive wing surfaces and AO^? posterior wing surfaces. A narrow band of AO⁺ cells extending proximodistally on both presumptive wing surfaces belongs to the posterior compartment. Two dorsal/ventral (dor./vent.) restrictions were found. The dor./vent. restriction equivalent to the dor./vent. border found in the adult wing was located at the ventral most edge of the AO-stained presumptive wing margin. A second restriction which was less strictly obeyed was found on the dorsal edge of the wing margin. We conclude that the whole presumptive wing margin is part of the dorsal compartment. Within the anterior wing margin an intensively stained oval was also found to be clonally restrictive. Therefore, territories were found within the prospective wing margin for which no such features have been identified in the adult Drosophila melanogaster wing.     

Isolation of a multigene family containing human alpha-tubulin sequences

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 base-pairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A · T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions.The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.     

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position ?30 and ?65 upstream from the structural genes, starting with a TATA box-like sequence. The 3′ flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Activation of a silent gene is accompanied by its demethylation

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.     

The effect of cupric ion on the reoxidation pathway of an IgG1 immunoglobulin

The addition of a 5–10μM of cupric ion to the reoxidation medium of a partially reduced IgG protein results in an altered pathway for covalent reassembly of the molecule. The formation of HL is favored over that of H₂ and H₂L.     

The polarity of axon growth in the wings of Drosophila melanogaster

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.     

Membrane adhesion in reconstituted proteoliposomes containing the light-harvesting chlorophyll ab-protein complex: The role of charged surface groups

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complexes (LHCP) of spinach, pea, and barley thylakoids apparently contain four nonidentical polypeptide subunits of between 29,000 and 23,000 daltons on highly resolving sodium dodecyl sulfate-polyacrylamide gradient gels. Trypsin treatment of the spinach complex degraded at least the two major subunits by approximately 2000 daltons and resulted in a three-subunit pattern on gels. Proteoliposomes reconstituted with LHCP and the chloroplast diacyl lipids aggregated markedly in the presence of cations but vesicles containing LHCP prepared from trypsin-treated thylakoids did not. Amino acid analysis of native- and trypsin-treated LHCP indicated that the fragment(s) released by trypsin, which is essential for cation-induced stacking of thylakoids, contains lysine and arginine, but not aspartate or glutamate, and is thus cationic. Carboxyl groups on the surface of LHCP were charge neutralized using a water-soluble carbodiimide (1-ethyl-(3-dimethylaminopropyl)carbodiimide) plus [¹⁴C]glycine ethyl ester. Only two or three sites were labeled per 26,000-dalton polypeptide equivalent and only a minor fraction of this (22–24%) was located in the surface fragment(s) released by trypsin. Both LHCP and LHCP proteoliposomes, after carboxyl modification, aggregated avidly at low salt concentrations. The findings suggest that exposed anionic groups on the surface of LHCP contribute to an electrostatic repulsive force between membranes which must be attenuated, either by cation binding or chemical neutralization, before membrane-membrane adhesion can occur. In line with this the binding of Mn²⁺ by LHCP (approximately four Mn²⁺ bound/26,000-dalton polypeptide equivalent) was sharply decreased after carboxyl modification.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.