A method for studying tissue specificity of maternally affected Drosophila melanogaster mutants: mosaic analysis of cinnamon.

Mosaic analysis is introduced to study tissue specificity of the maternal effect which characterizes the wild-type allele of cinnamon (=cin). The methodology presented is applicable to studies of other maternal effect mutations, as well as to female or male sterility mutations. One hundred and forty-three pal-induced fertile cin-mosaic females were obtained, and the degree to which they were capable of maternally affecting their homozygous cin daughters was correlated to their cuticular and germinal genetic constitution. From this analysis the following conclusions are drawn: (1) The presence of a wild-type (cin⁺) allele in maternal germ cells constitutes a sufficient condition for the full expression of the cin⁺ characteristic maternal effect: All cin offspring derived from such cells have normal viability and eye color. (2) This effect is confined to the descendants of a particular germ cell and does not extend to the descendants of other non-cin⁺ germ cells in the same or in a neighboring ovary. (3) A wild-type allele in a germ cell constitutes a necessary condition for the eye color maternal effect. (4) When a maternal germ line is wholly mutant, nonmutant constitution of an additional focus may result in rescue of more than half of the homozygous cin offspring (all with mutant eye color). Mosaic analysis suggests that this somatic viability focus originates from the posterior region of the blastoderm. These conclusions were tested and confirmed by transplanting heterozygous cin ovaries, wild-type Malphigian tubes, and wild-type fat body into homozygous cin hosts. In addition, transplantations of homozygous cin ovaries into wild-type hosts suggest that the posterior maternal viability focus corresponds to the mesodermal components of the ovaries.     

Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster

Agametic, a maternal-effect mutation, causes the absence of germ cells in approximately 40% of the gonads of flies derived from homozygous females. The nature of the deficiency in the eggs produced by these flies was examined. Ultrastructural abnormalities were seen in the polar granules of some eggs shortly after fertilization. Although a normal number of pole cells form, some are abnormal with degenerating polar granules and nuclear bodies and they contain myeloid bodies. The pole cells reach the gonads and at 14 hr of development all the gonads contain germ cells. However, in 40% of the gonads the germ cells become necrotic and disappear. Thus, the source of agametic gonads in the adult is embryonic death of pole cells in some gonads. To test whether this gonadal death is an autonomous deficiency of the mutant pole cells, mosaic pole cell populations were produced by reciprocal pole cell transplantation. In both types of transplants, the mutant pole cells died autonomously. In eight instances gonads containing only donor pole cells were obtained. Since mutant pole cells die when wild-type pole cells normally begin dividing, we suggest that the lesion affects the ability of these mutant pole cells to reenter the cell cycle.     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

Regulative interactions between cells of wing discs from different dipteran species

The mechanism by which patterns are produced appears to be repeated in each segment of an animal, and it has been proposed that it may even have been conserved in evolution so that different species would have the same system of positional information. This idea has been tested by mixing cells of a defined fragment of the wing disc of Drosophila melanogaster with wing disc fragments of five other dipteran species to assay the ability of these disc fragments to stimulate intercalary regeneration of the D. melanogaster cells. The genetically marked (y; mwh) D. melanogaster fragment was mechanically mixed with wing discs or wing disc fragments of four drosophilids (D. melanogaster as a control, D. virilis, D. hydei, Zaprionus vittiger), of Musca domestica, and of Piophila casei. The mixed aggregates were cultured in vivo for 7 days, then metamorphosed in D. melanogaster larval hosts. The D. melanogaster fragments were only stimulated to regenerate when combined with complementary fragments from D. melanogaster or D. virilis wing discs. In the combination between D. melanogaster and D. hydei, the tissue formed integrated mosaic patterns, but no regeneration ensued. The one positive result (D. melanogaster mixed with D. virilis) shows that positional cues can be exchanged and correctly interpreted between cells of different species. The negative results do not prove that the mechanism for establishing patterns is different in the tested species, but may be due to incompatibilities that are not related to pattern formation.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Heterogeneity of the ribosomal genes in mice and men

The structures of mouse and human ribosomal DNA were studied using the restriction enzymes Eco R1 and Hind III. Individual mice or humans showed a heterogeneous pattern of restriction fragments resulting from differences in the non-transcribed spacer DNA. Six individual mice from the inbred strain CBA/H-T6 had identical patterns. The same pattern was shown by another CBA strain and by C3H. These strains were originally derived from a BALB X DBA cross made in 1920. Different patterns were found for BALB/c, C57BL and Mus poschiavinus. Cultured cells derived from C3H mice (L cells) showed a pattern quantitatively different from that of the parent strain, but two myeloma cell lines derived from BALB/c showed the same pattern as BALB/c mice. Ribosomal DNA in man is also heterogeneous. Differences were observed between human DNAs in the amounts of the different spacer classes. Studies on mouse-human cell hybrids suggest that some spacer classes are present on more than one of the five human nucleolus organizers.     

Regeneration of symmetrical forelimbs in the axolotl,Ambystoma mexicanum

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).     

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l-3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K_i is 100 μm. It was shown that l-3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l-3,4-[¹⁴C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l-prolyl-tRNA. The addition of l-3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.     

The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer

The hyaline layer (HL) surrounding the sea urchin blastula appears to dissolve in 1 M glycine. However, after this treatment, there persists over the surfaces of the blastomeres a layer of material, referred to here as the apical lamina (AL), that sloughs off as an adhesive convoluted bag upon gradual dissociation of the embryo. Isolated hyaline layers, referred to as HL-AL complexes, were analyzed by urea-SDS-polyacrylamide gel electrophoresis. A major protein of the HL-AL complex, hyalin, bands or precipitates in the stacking gel. Two other major proteins, both strongly PAS positive, migrate with apparent molecular weights of 175K and 145K daltons. As with intact embryos, the glycine wash removes the hyalin protein from the isolated HL-AL complex, leaving the undissolved AL which consists primarily of the 175K- and 145K-dalton proteins. The embryo's own perivitelline-localized cortical granule peroxidase heavily radioiodinates the proteins of the HL-AL complex, further verifying their apical, extracellular location. Unlike hyalin, the AL proteins do not precipitate with calcium ions. Compared to the entire HL-AL complex, the AL contains a greater percentage of carbohydrate. No sialic acid is associated with the HL-AL complex, but the AL contains some sulfate. In contrast to a published report based on ultrastructural staining, no biochemical evidence was found in this study for the presence of collagen or significant glycosaminoglycan within the HL-AL complex. No developmental differences were observed in AL proteins from 1-hr-old embryos compared to those from blastulae. However, there is evidence suggesting heterogeneity and developmental differences in hyalin. The possible organization of hyalin and the AL proteins into separate layers surrounding the embryo is discussed. The influence of the AL proteins in morphogenesis and cell adhesion is considered, and hypothetical roles attributed to the HL and hyalin are critically questioned.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

The digital pads of the tree frog, Hyla cinerea. I. The epidermis

As the digital pad cells grow from the germinal epithelium, the desmosomes on the distal surface oF tile cells become aligned and form pegs filled with tonofilaments on the surface of the outer row of cells. The outer pad cells are separated from each other distally, thus the cell and the pegs form two sizes of protrusions in series on the pad surface which can fit into substratum/irregularities. A dense material, apparently derived from transforming bodies in the cells, coats the plasma membrane of the surface cells, presumably to strengthen them.     

The digital pads of the tree frog, Hyla cinerea. II. The mucous glands

The mucous glands consist of a single row of cells surrounded by smooth muscle. The cells are attached at their apical and basal regions and only cytoplasmic projections loosely link the lateral aspects of adjacent cells. Material accumulates in the cisternae of the rough endoplasmic reticulum, appears to form into dense granules in the Golgi apparatus, and then before being secreted, undergoes chemical and morphological alterations. Since some glands secrete onto the dorsal epidermis of the digits, the mucous is believed to function as a wetting agent for the skin as well as an aid to climbing.     

Thermal elution chromatography and the resolution of nucleic acids on hydroxylapatite

Thermal elution chromatography of nucleic acids on hydroxylapatite was studied from a technical standpoint. It is shown that current methods for selecting elution buffers are inadequate. The construction of window diagrams for the purpose of determining suitable conditions is demonstrated. The resolving ability of various buffer-hydroxylapatite systems was studied in some detail. The best system for resolving single- from double-stranded nucleic acids was found to be the use of potassium phosphate together with Bio-Rad HTP (non-DNA grade) which has been preheated in phosphate buffer. Sodium phosphate gives the best resolution among various species of double-stranded nucleic acid.     

Tubulin synthesis during ciliogenesis in the mouse oviduct.

We reported earlier that tubulin levels increase in the developing mouse oviduct during that period after birth when ciliogenesis is at a maximum (Staprans, I., and Dirksen, E. R. (1974) J. Cell Biol., 62, 164). To determine the degree to which de novo synthesis and tubulin pools contribute to this increase, [³H]leucine-incorporation experiments were performed in vivo and in culture. Soluble, particulate and axonemal fractions, obtained from homogenized oviducts of 3-, 5-, 8- and 12-day-old suckling mice, were electrophoresed on sodium dodecyl sulfate gels and the specific activity of the tubulin band determined. The present work shows that more than 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts is synthesized de novo. From both the in vivo and in culture experiments we conclude that although tubulin pools are present in mouse oviduct, they are continuously being replenished by newly synthesized protein as there is a rapid outflow from the soluble and particulate to the axonemal fraction into structures such as basal bodies and cilia. This burst of de novo tubulin synthesis corresponds to evidence from electron microscopic autoradiography, where label is present to a greater extent over centriole precursors and basal bodies than over other cell organelles. [³H]leucine incorporation into tubulin was inhibited by cycloheximide, demonstrating that we are dealing with synthesis, while colchicine below 10^?3, M concentration had no effect on tubulin assembly into axonemes.     

Proteins synthesized in lymphoid cells early after activation by phytohemagglutinin

A double-isotope labeling approach has been employed in an attempt to identify the proteins synthesized by lymphocytes early after stimulation by phytohemagglutinin (PHA). The earliest effect of PHA, within the first hour, was the induction of large aggregates of cellular proteins, which were not dissociated by 1% sodium dodecyl sulfate (SDS) in the absence of β-mercaptoethanol. These aggregates were composed of proteins of molecular weight approximately 70,000, but they did not include PHA. The aggregates were made up of preexisting as well as newly synthesized cellular proteins. Subsequently, within the first 2 hr after the addition of PHA, there was a nonspecific stimulation of protein synthesis. This was followed by the preferential synthesis of several classes of proteins including at least one group of nuclear proteins. The structural changes described here are among the earliest events known to occur within the lymphoid cell after its interaction with PHA.     

Intercellular adhesion in the cellular slime mold Polysphondylium pallidum inhibited by interaction of asialofetuin or specific univalent antibody with endogenous cell surface lectin.

Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.     

Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle

Isoforms of C-protein in adult chickens which differ in fast (pectoralis major, PM) and slow (anterior latissimus dorsi, ALD) skeletal muscles can be distinguished immunochemically with monoclonal antibodies (McAbs) specific for the respective fast (MF-1) and slow (ALD-66) protein variants (Reinach et al., 1982 and 1983). The expression of these C-proteins during chick muscle development in vivo has been analyzed by immunoblot and immunofluorescence procedures. Neither MF-1 nor ALD-66 reacted with whole-cell lysates or myofibrils from PM of 12-day-old embryos. However, both McAbs bound to peptides of 145 kDa in PM from late embryonic and young posthatched chickens. All of the myofibers in these muscles reacted with both antibodies, but the binding of the anti-slow McAb (ALD-66) diminished progressively with age and was completely negative with PM by 2 weeks after hatching. In contrast, the ALD muscle from 17 days in ovo thru adulthood only reacted with ALD-66; no binding of MF-1 could be detected at these stages. Since both fast and slow myosin light chains (LC) coexist within embryonic pectoralis and ALD muscles (e.g., G. F. Gauthier, S. Lowey, P. A. Benfield, and A. W. Hobbs, 1982, J. Cell Biol.92, 471–484) yet segregate to specific fast and slow muscle fibers at different stages of development, the temporal transitions of C-protein and myosin LC were compared during myogenesis. “Slow-type” C-protein appeared after the disappearance of slow myosin light chains, whereas the accumulation of the “fast-type” light chains occurred before the expression of “fast-type” C-protein. The pattern of isoform transitions appears to be far more complex than previously suspected.     

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.