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The interactions of genes and environment during development have been fascinating, controversial subjects for more than a century. One of the earliest reports of environmental effects on insect development was made by George Dorfmeister in U 1854. He demonstrated that exposure to extreme heat or cold could change the pattern on the butterfly’s wings. More recent studies on environmental effects on insect development have primarily been done in fruit flies. Drosophila is used as a model system for studying developmental genetics because of its short life cycle and small genome size. These same attributes also make it a good model system for studying environmental effects on gene expression during development. Goldschmidt was the first to extensively study environmental influences on development in Drosophila. He showed that many different developmental defects which resemble mutant defects can be induced by heating during the pupal period. Unlike mutations, however, these defects are not inherited. Some examples of heat induced defects compared to mutant defects are shown in Fig. 1. Goldschmidt coined the name “phenocopy” to describe environmentally induced developmental defects. This name was chosen in order to emphasize the resemblance of the environmentally induced defects to mutant phenotypes (Goldschmidt 1935). Goldschmidt was impressed by the fact that chemicals as well as heat induced the same types of defects if they were given during the same sensitive period. concluded that mutations, chemicals, and heat, might all affect a different initial chemical process, but the end result was the same because they diverted development from a normal path onto an abnormal path, and he felt that there are a limited number of abnormal developmental pathways which are not lethal.

The construction of cell hairs (trichomes) on the wings of Drosophila occurs in synchrony on 30,000 cells over a period of about 20 hr. Changes in both morphology and patterns of protein synthesis occur rapidly during this time period. In this report we describe the use of stress-induced (heat shock) abnormalities in morphogenesis to provide further details on the stepwise processes of differentiation within single wing cells. A cartoon summary of the overall process and a discussion of some possible mechanisms is included.

The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [35S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified. Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [3H]uridine labelling. By correlating the amount of [3H]uridine in some puffs with the level of [35S]methionine in some bands a tentative relation is suggested in a few instances. The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.

Heat shock uncovers the recessive forked phenotype when heterozygotes between f36a and wild-type are heated during sensitive periods in pupal development. We call the phenocopy of a mutant in such a heterozygote a heterocopy. The heterocopy in f36a/+ is virtually identical to the mutant phenotype; however, bristles on different parts of the body are affected during different sensitive periods. We discuss the hypothesis that the heat shock acts by affecting expression of the wild-type gene product corresponding to the mutant gene. The sensitive period for heterocopy induction in a specific tissue is proposed to correspond to the normal time of gene expression for the forked gene product in a particular tissue.

The 84,000 dalton heat shock protein from Drosophila melanogaster has been purified by ammonium sulphate fractionation, hydroxyapatite chromatography, and gel electrophoresis. An antiserum directed against this protein has been prepared. Indirect immunoprecipitation experiments using this antiserum have shown that this protein is synthesized under normal conditions in at least three different tissues representing animals at different developmental stages, including salivary glands from larvae and prepupae.

Phenocopies are developmental defects induced by environmental treatments during differentiation. Because of their resemblance to mutant phenotypes it has been suggested that phenocopies are due to environmental effects on the expression of specific genes during development. In this paper we describe the heat shock (40.8 degrees C) induction of a multiple wing hair phenocopy in the mutant heterozygote (mwh/+). The mwh phenocopy is only induced in heterozygotes of the recessive mutant during a short sensitive period which appears to be the time of expression of the multiple wing hair gene. We suggest that this phenocopy is due to failure of mwh gene expression and that phenocopy sensitive periods may be useful in identifying expression periods for particular genes during development. Furthermore we have been able to demonstrate that a 35 degrees C pretreatment will prevent the induction of the multiple wing hair phenocopy. A similar 35 degrees C pretreatment prevents induction of several different phenocopies by heat in wild-type flies (N. S. Petersen and H. K. Mitchell (1985). In "Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. X, Biochemistry." Pergamon, New York). This indicates a common molecular mechanism for both the induction and the prevention of heat-induced phenocopies.

Mature larvae of Drosophila busckii have been found to contain high concentrations of a tyrosine derivative which differs from tyrosine-O-phosphate in its chromatographic and electrophoretic properties as well as its elution profile from the column of an amino acid analyzer. Procedures for the isolation and purification of this compound are described. The availabledata all indicate that it is β-glucosyl-O-tyrosine. Its rapid accumulation in the late third instar larvae and its sudden disappearance at pupariation suggest that the glucoside serves as a tyrosine reservoir for the sclerotization of the pupal case.

We describe in this paper details of morphogenesis of wing hairs in Drosophila pupae. The ultimate objective is to relate specific protein components used in hair construction to specific components produced in the rapidly changing patterns of gene expression that are characteristic for the period of hair differentiation in wing cells ( H. K. Mitchell and N. S. Petersen, 1981 , Dev. Biol. 85, 233–242). Hair extrusion to essentially full size occurs quite suddenly at about 34 hr (postpupariation) and this is followed by deposition of a double-layer of cuticulin during the next 4 to 5 hr. Extreme changes in shape of cells and hairs, probably related to actin synthesis, then occur for the next 5 to 6 hr. Deposition of fibers within the hairs and on hair pedestals follows. Formation of cuticle on the cell surface begins and continues until some time in the 60-hr range. It appears that cuticle is formed only on the cell surface and not in hairs or on the top of hair pedestals. The protein synthesis patterns associated with these events are described.

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5-20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.

A mild heat shock at 35 degrees C, which induces heat shock gene expression, greatly enhances survival and the recovery of protein synthesis in Drosophila cells after a higher temperature heat shock. The 35 degrees C treatment is also effective in preventing heat-induced developmental defects in pupae. We show here that the major larval mRNAs are present in approximately normal (25 degrees C) concentrations after a 40.1 degrees C heat shock whether or not the animals receive a pretreatment. This indicates that the pretreatment affects translation directly rather than messenger concentration. We also observe selective translation of heat shock messages and some 25 degrees C messages during recovery from heat shock.

Transcriptional changes with development of the imaginal thoracic hypoderm of Drosophila melanogaster were evaluated by studies on pulse labeling and total composition with respect to protein. Evidence presented indicates that certain glycoproteins produced by prepupal salivary glands are important for bristle development long after the salivary gland is histolyzed. Striking changes in protein synthesis with development can be correlated with major hypodermal functions such as bristle formation, chitin deposition, and a moltlike process which occurs around 52 hr after puparium formation.

Differentiation of the single cells that yield bristles and hairs in Drosophila has been examined by studies of phenocopies induced by heat shock. Phenocopies that resemble the mutants hook and javelin are produced by application of the stress within narrow and specific time ranges during metamorphosis. Double phenocopies can be induced by successive heat shocks at appropriate time intervals. Phenocopy production may be related to heat shock effects on RNA synthesis through a feedback inhibition of transcription by heat shock proteins.

Experiments on short-term and pulse-chase labeling of chromosome proteins of the salivary glands of Drosophila melanogaster show unique patterns of label in the vicinity of chromosome puffs. A high turnover rate is indicated for these nonhistone proteins, which appear to form a fibrous sheath around the chromosomes. Acrylamide gel analyses of the chromosomal proteins that are quickly labeled, comparing compositions at different stages of development with compositions after heat shock, show that all are different and dependent on which chromosomal puffs are active and producing messenger RNA. The necessity for a continuous and rapid interchange of protein between the nucleus and cytoplasm is indicated, and it appears that regulation of gene activity must be related to this dynamic state of protein exchange. From the technical standpoint, it has been found that scanning electron microscopy (SEM) is especially useful for observing silver grains on opaque autoradiographs. It appears also that SEM will prove useful in a variety of studies of chromosome structure.

Extreme and rapid changes in the synthesis of messenger RNAs and proteins accompany differentiation in wing tissues of Drosophila. Of the six actin genes, at least three are expressed in wing cells, some during the most extreme changes in cell shape. However, different messages of the set appear, decay, and reappear on a regulated temporal program. These results show that actin expression is stage-specific in a single cell type.

Very short heat shocks are administered to carefully staged early embryos of Drosophila melanogaster, and the effects on protein synthesis pattern investigated. A shock as short as 2 min will induce the heat shock response (reduction of normal protein synthesis, increased synthesis of the heat shock proteins) in syncytial blastoderm or later stages. Thus the initial events of the heat shock response must occur within 2 min, and not reverse upon rapid return to 22 degrees C. A low level of synthesis of the 70 kDA heat shock protein is sometimes visible in unshocked animals, but may be induced by the labeling procedure. Survival following a short shock is not strictly correlated with a high level of heat shock response. Pre-blastoderm embryos do not produce significant heat shock protein, but survive a 2 min 43 degrees C heat shock better than do heat shock response competent blastoderm embryos. The protein synthesis pattern prior to the blastoderm stage is very stable, possibly enhancing survival following a short shock. Shocks of 3 min or longer are more detrimental to pre-blastoderm embryos than to later stages, confirming the role of the heat shock response in survival following a longer shock. Stage-specific developmental defects (phenocopies) may be induced by heat shock at the blastoderm or later stages. Induction of these defects may require disruption of the normal protein synthesis pattern. Use of very short heat shocks to induce the heat shock response will be valuable in identifying the precise time at which a specific defect can be induced.

Before both larval moults in Drosophila melanogaster , the chitin in the cuticle is digested to a significant degree by the moulting fluid. A spurt of chitinase activity appears just before each ecdysis, drops sharply after the first ecdysis, and begins to rise again just about the time that chitin degradation becomes evident. The level of enzyme activity/mg of soluble protein reached just before the second ecdysis is about twice that reached before the first, and this declines gradually after the ecdysis until puparium formation. Chitinase activity is measured with a viscometric assay on a chitosan substrate. The enzyme activity is stable, with no loosely bound cofactor. Data also exist supporting the presence of more than one enzyme fraction in Drosophila with chitinase activity.

P. H. Lowy , H. K. Mitchell and U. W. Tracy . Bee venom phospholipase A 2 : effects on Drosophila larvae, HeLa cells and membranes. Toxicon 14, 203–212, 1976.— Drosophila larvae injected with sublethal doses of bee venom phospholipase A 2 become lethargic, stop feeding and growing, but undergo metamorphosis and emerge as miniature flies which give rise to normal progeny. One-third of the electrophoretically homogeneous enzyme binds more strongly than the rest to membranes from larval tissue. The enzyme increases the permeability of the inner membrance of pigeon heart mitochondria, allowing passage of neutral molecules at least as large as tetrasaccharide. Electron microscope evidence shows a concentration-dependent swelling short of actual membrane disruption even at very high enzyme concentrations. The enzyme does not affect erythrocytes but causes the appearance of many lipid droplets in the cytoplasm of HeLa cells. The peculiar starvation-like effect of phospholipase A 2 on larvae may result from leakage due to conformational changes in membranes rather than extensive hydrolysis of membrane phospholipid.

We have for some years been making use of phenocopies in Drosophila, as induced by heat shock, as tools for studies of the molecular events in morphogenesis [18, 21, 22]. In this paper, we have brought together some accumulated information on the conditions for phenocopy production, on a temporal sequence of sensitivity to induction, and on the nature of many of the morphogenetic abnormalities that can be induced. In general, the induction of phenocopies by heat shock requires conditions drastic enough to turn off transcriptional activities but not extreme enough to prevent recovery. This situation is most easily achieved in pupal stages where heat resistance is high, but even in this range, resistance varies with the stage of development. The phenocopies described resemble, for the most part, mutants that affect structures derived from epithelial differentiation or muscle development.

The 70-kDa heat shock protein of Drosophila decays in vivo at a much faster rate than other abundantly labeled proteins. Degradation also occurs in vitro, even during electrophoresis. It appears that this degradation is not mediated by a general protease and that the 70-kDa heat shock protein has a slow proteolytic action upon itself. Heat-induced proteins in CHO cells and a mouse cell line also degrade spontaneously in vitro, as do certain non-heat shock proteins from Drosophila tissues as well as the cell lines.

Our earlier evidence concerning the complexity of the activation process for Drosophila phenol oxidase has been confirmed by separation and purification of six proteins involved. This is a minimal number required in a reaction series or cascade to yield active enzyme, and at least two more proteins are known to be involved. A simpler system involving only the last step with one precursor and one activation as has been reported in the literature is consistent with the cascade picture, but the whole complex system must be considered when dealing with genetic and developmental regulation of pigmentation and sclerotization. Details are given for partial or complete purification of six of the proteins involved and evidence for their modes of interaction is presented.

Autoproteolysis is an essential activity in the expression of the entire genomes of a number of viruses. That is, new viruses can be produced only after large polyprotein products translated from the genome or from subgenomic mRNA degrade themselves to the polypeptides necessary for RNA replication or for the construction of new virus particles. We have recently shown that the major heat shock protein of Drosophila and a mouse cell line (70 kDa) also undergoes autoproteolysis with the production of specific patterns of smaller polypeptides. We show now that many other proteins in eucaryotic tissues also have a potential for self-degradation. We suggest that special coding regions in many genes may have important roles in both protein turnover and in the production of regulatory peptides.

Pupae of Drosophila melanogaster were heat-shocked under conditions required to induce phenocopies in more than 90% of the flies that subsequently emerge. The effects of these treatments on protein synthesis in two tissues (thoracic epithelium and brain) were followed for several hours after the heat treatments. Results from pulse-labeling and protein separations on sodium dodecylsulfate (SDS) acrylamide gels showed a virtually complete cessation of protein synthesis immediately after the shock, followed by a noncoordinate resumption of the starting pattern. Similar experiments following double heat shocks demonstrated a more rapid resumption of synthesis of heat shock proteins after two successive heat treatments than after a single one.

Mild heat treatments applied to whole animals or cell cultures of Drosophila prior to lethal heat shocks result in increased survival and protection against phenocopy induction. The optimal condition for the preliminary mild heat treatment is that which induces the synthesis of heat-shock proteins but does not turn off the protein synthesis that is in progress. Recovery of protein synthesis but not RNA synthesis following a drastic heat shock is much enhanced by the pretreatments. The results suggest that the protection for survival and against phenocopy induction is due to storage of messenger RNA.

A procedure is described for extraction and fractionation of RNA components from microgram quantities of tissues. Illustrations are from experiments with salivary glands, brains, and whole animals of Drosophila melanogaster. The procedures given are suitable for studies of changes in RNA components as related to development.

We describe variants of three heat-shock proteins of Drosophilg melanogaster and their use to map the chromosome regions that contain the coding sequences for these proteins. All three map to a region on chromosome 3L that includes only one heat-shock puff, designated as 67B. The results imply that the genes coding for at least three heat-shock proteins are included within the 67B region.

THE SECOND paper of this series (Beadle, 1945b) presents some details on the production and isolation of a number of mutants of Neurospora. In most cases investigated, the new strains differ from the wild type parent by a single gene and this gene is normally concerned with a step in the sequence of reactions leading to the production of an essential growth substance. It is considered that the gene controls the production of a specific enzyme that in turn catalyzes a specific reaction. As a rule, the mutations obtained in Neurospora appear to involve a complete inactivation or an actual physical deficiency in one gene, but recent studies on a group of temperature-sensitive mutants suggest that intermediate modifications of genes also exist. Oiily one mutant has been found that requires the vitamin riboflavin (isolation number 51602), and this requirement is a function of the temperature at which the mold is allowed to grow. That is, it grows and synthesizes riboflavin at a rate approaching that of a wild type strain at temperatures below 250C. while an external source of the vitamin is necessary above 28?C. The present paper is concerned with the inheritance of this mutant type and its growth and its temperature characteristics as well as some consideration of gene action in riboflavin synthesis in Neurospora. GENETIC ANALYSIS.-Mutant 51602 was crossed to wild type and the eight spores from each of twenty asci were isolated in order. In all twenty cases the temperature-sensitive, riboflavin-requiring mutant was recovered from four spores and wild type from the other four. Crosses to four mutants known to differ from wild type by a single gene gave similar results, and here too the mutant was recovered intact in its temperature-sensitivitv and nutritional requirements from four of the spores in each of 80 asci. Therefore, it may be concluded that mutant 51602 differs from wild type by a single gene. Experiments on linkage will be reported in a general paper on this subject. GROWTH.-Experiments on growth were carried out using the same general technique that has been extensively applied in this laboratory (Horowitz and Beadle, 1943; Beadle, 1944). Twenty ml. of medium contained in 125 ml. Erlenmeyer flasks were inoculated with a dilute conidial suspension and the mold allowed to grow at designated tem1Received for publication September 10, 1945. This work was supported by funds supplied by the Nutrition Foundation and the Rockefeller Foundation. The compounds lumichrome, lumiflavin, 3,4-dimethylphenyl-d, l'-ribamine and 2-amino-4, 5 dimethyphenyl-d, 1'-ribamine were obtainedthrough the courtesy of Drs. Jackson W. Foster and Max Tishler of Merck and Company. Riboflavin-adenine dinucleotide was generously furnished, from a very small supply, by Dr. F. M. Strong of the University of Wisconsin. peratures and periods of time. The mycelial pads were then removed, pressed on filter paper, placed in order on cardboard or filter paper, dried for three hours at 850C., cooled and finally weighed. The medium, containing inorganic salts, sucrose, biotin and water, has been described a number of times (Horowitz and Beadle, 1943; Beadle, 1945b). The appropriate supplement required to proiduce growth of the mutant was added in 2 ml. or less of water. At 310C. mutant 51602 does not grow without addition of riboflavin to the minimal medium that supports growth for wild type Neurospora. With increasing quantities of added riboflavin an Sshaped growth curve is obtained. The range of concentration varies with the temperature but extends from approximately 0.1 to 2.5 micrograms of riboflavin. Growth curves for various temperatures can be determined from the data represented in figure 1.

Uricase from Neurospora crassa has been purified 400-fold. The properties of this enzyme are similar to those of the animal uricases except that it is more soluble at low pH values. Addition of uric acid to the growth medium causes a twofold increase in the amount of enzyme. One atom of oxygen is consumed per mole of uric acid decomposed in the presence of catalase. Spectra taken during the course of the reaction have confirmed the presence of ultraviolet-absorbing intermediates.

In Drosophila recessive mutations have been observed which suppress the effect of specific mutations at other loci. Thus an individual, homozygous for a suppressor gene in the mutant form and for the mutant gene upon which the suppressor acts, is phenotypically wild type, or nearly so. Suppressors of black,(1) purple,(2,3) sable(4) and vermilion(4, 5) are among those, which have been reported. In the vermilion case there is evidence(5) which shows that the action of the suppressor is to restore v+ substance (kynurenine), the formation of which is prevented when the mutant gene vermilion is present in the homozygous condition.

Procedures are described for obtaining phytoene and each of the carotenoid pigments of Neurospora crassa spectroscopically and radiochemically pure when 2-14C-mevalonate is used as a labeled precursor. Using these techniques, 14C-label was shown to be incorporated into phytoene, zeta-carotene, neurosporene, beta-zeacarotene, gamma-carotene, lycopene, and 3,4-dehydrolycopene. The carotenoid beta-zeacarotene, which had not previously been reported to be synthesized by Neurospora crassa, was shown to accumulate under certain culture conditions. Labeling and time-course data are presented which support the proposal that the carotenoids are all synthesized from phytoene by a series of sequential reactions.

Of the five protein components that have been shown to be involved in phenol oxidase production in extracts of Drosophila melanogaster, component S is rate limiting in the activation process. By means of sectioning of whole animals and dissection of specific tissues it has been established that S is initially localized in the larval salivary gland where it is apparently produced and secreted. The S component appears to be released in the haemolymph in late third-instar larvae but it is not present in an active form in early prepupae. A procedure is given for the purification of and the measurement of the activity of the S component.

An interaction of fatty acids with horse heart cytochrome c which produces chemical, spectral, and electrophoretic changes in the protein was investigated. Cytochrome c in the presence of fatty acids became much more susceptible to destructive oxidation by hydrogen peroxide, was no longer readily reduced by ascorbic acid, and when treated in the reduced form could be oxidized directly by molecular oxygen. It was concluded that these and other changes in the properties of cytochrome c brought about by fatty acids were probably associated with a partial unfolding of the protein moiety of the cytochrome molecule from around its planar heme. At a concentration of 1.0 m M , palmitic acid was the most effective of the four saturated fatty acids tried, indicating an apparent specificity between fatty acids and cytochrome c.

The discovery of orotic acid (1,2-carboxyuracil) in cow's milk by Biscaro and Belloni (1), followed by its identification and synthesis (2-4), led to a number of speculations as to its biological origin and significance (,3,5,6). A definite connection of orotic acid with the biosynthesis of nucleic acid pyrimidines is provided by the finding that orotic acid (7) as well as thymine (8,9) can supplement or replace the folic acid required by certain microorganisms. As suggested by Chattaway (7), it would appear that folic acid has a function in the biosynthesis of pyrimidines. Furthermore, this function is probably concerned in some step prior to the appearance of orotic acid in the biosynthetic series. More recently it was shown by Loring and Pierce (10) that orotic acid could be substituted for uracil in satisfying the growth requirements of some pyrimidineless mutants of the mold Neurospora. Investigations of orotic acid in this laboratory have led to a new method of synthesis of the compound (11) and to some suggestions concerning its relation to the biosynthesis of nucleic acids in Neurospora (12). The results of the present work are in accord with the previous suggestions and provide further evidence on the biological origin and function of orotic acid.

A method is described for measuring the radioactivity of proteins labeled with C 14 or H 3 following their separation by disk electrophoresis. After the water in the gel has been replaced with a toluene-based scintillator solution, the radioactivity is measured directly in the acrylamide gel with scintillation techniques. The counting efficiency for C 14 in this system is 81 ± 12%, whereas that for H 3 is about 23%. With the use of a simple soaking apparatus it is possible to prepare 74 gel slices, with a minimum of handling, in 9 to 18 hr, depending upon the size of the slices.

The principal aim of the present report is to present a series of data which demonstrate the applicability of simple chromatographic methods to the problems of investigating biochemical changes which occur during development of Drosophila melanogaster and studying biochemical differences between mutants of this organism. Extensive analyses of the data and their significance in relation to the work of other investigators will be reserved, for the most part, for subsequent and more detailed presentations.

A bacteriophage (Tφ3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60 C, the optimal growth temperature of the host. At 60 C, the phage had a latent period of 18 min and a burst size of about 200. The phage was comparatively thermostable in broth. The halflife of Tφ3 was 400 min at 60 C, 120 min at 65 C, 40 min at 70 C, and 12 min at 75 C. The activation energy for the heat inactivation of Tφ3 was 56,000 cal. The buoyant density of Tφ3 in a cesium chloride density gradient was 1.526 g/ml. Electron micrographs of Tφ3 indicate that the phage has a head that is 57 mμ long. The dimensions and shape of the head are compatible with those of a regular icosahedron. Each edge of the head is 29 mμ long. The tail of Tφ3 is 125 mμ long and 10 mμ wide. There are about 30 cross-striations that are spaced at 3.9-mμ intervals along the tail. Under the conditions investigated, Tφ3 adsorbed slowly to the host. Only 2.8% of the phage adsorbed in 10 min at 60 C, the normal incubation temperature that was used. Tφ3 was not infective to four other thermophilic strains or to two mesophilic strains of bacteria.

Experiments by a number of workers have established the fact that addition of tryptophan to the diet results in increased synthesis of nicotinic acid by a number of mammals.

The role of tyrosine-O-phosphate as a substrate for tanning of the pupal case of Drosophila has been examined. From 14C tyrosine label experiments combined with studies on dopa decarboxylase it appears likely that tyrosine-O-phosphate is hydrolyzed prior to participation in the tanning reaction. Subsequent metabolism may be through dopamine and N-acetyl-dopamine, as has been described for Calliphora, although these intermediates are not accumulated in Drosophila. On this basis the tyrosine phosphate may represent only a relatively inert tyrosine reservoir. However, other functions such as a role in transport remain as possibilities.

In 1936 Richardson (1) showed that uracil was essential for the anaerobic growth of Staphylococcus aureus, but not for aerobic growth of the same organism. Of five strains tested three required uracil, while one required both guanine and uracil for growth. Thymine or cytosine did not replace uracil for this organism. These experiments suggested that hydrolytic products of nucleic acids might become factors limiting growth of various organisms under certain conditions. Bonner and Haagen-Smit (2) in 1939 showed that adenine greatly stimulated growth of leaves under defined conditions, while Moller (3) showed that adenine was required for growth of Streptobacterium plantarum. Pappenheimer and Hottle (4) recently showed that adenine was necessary for the growth of a strain of Group A hemolytic streptococci; it could be replaced by hypoxanthine, guanine, anthine, guanylic acid or adenylic acid. They made the very interesting observation that adenine was unnecessary for growth of this organism if the carbon dioxide tension was maintained at a sufficiently high level.

It was shown recently that the poky strain of Neurospora accumulates large amounts of cytochrome c, but is deficient in cytochromes a and b and thus in cytochrome oxidase and succinic acid oxidase activities (1). These findings, which suggest that respiration in poky does not utilize the cytochrome system, led to a study of the respiratory system of poky compared with that of wild type. The experiments reported here are concerned with the effects of the inhibitors cyanide and azide on the respiration of intact mycelium of poky and wild type, the oxygen uptake of cell-free extracts in the oxidation of their endogenous substrates, the cofactor requirement for this oxidation, and the effects of azide and of oxygen tension. Evidence presented confirms that, in poky, respiration does not depend upon the cytochromes, but upon another terminal oxidase system in which flavin-adenine dinucleotide (FAD) enzymes appear to be concerned.

In view of the widespread occurrence of the amino acid-containing lipides and the unique course of their metabolism during development of Drosophila, we have carried out extensive investigations concerned with their isolation and chemical nature. The present report is concerned primarily with techniques and procedures developed to insure removal of non-lipide contaminants from preparations of these lipides.

Modern biochemistry has so far encountered no phylogenetic boundaries in terms of fundamental principles of molecular biology and, in this sense, it is archaic to review only Drosophila biochemistry. However, operationally Drosophila occupies a unique position as biological material useful for the solution of general problems because of the large numbers of mutants and species available, the favorable life cycle and culture conditions, and the very extensive literature describing chromosomes and chromosomal manipulations. That increasingly active exploitation of this large reservoir of material and information is in progress, is evident from recent literature and, in general, only a selected portion of the most recent literature is covered here. Particular emphasis is given to biochemical aspects having to do with the nature of gene functions and gene products.