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Concerted Gene Expressions in Elicited Fibroin Synthesis
Abstract
Our studies show that the making of fibroin by mechanically stimulated spider glands seems to require a series of well-orchestrated gene expressions. Monitoring of the process through time sequence has revealed four transient waves of molecular syntheses. The last and most dramatic of these events is the synthesis of the full-size fibroin product, which is preceded by a wave which generates template RNA by a 60-min interval. The other two events generate small RNAs. Analyses of the first of the small RNA-generating bouts, consistently of higher magnitude than the subsequent one, displays upgrading of 5S RNA, to a higher extent of Ul snRNA, and a dramatic boost in alanine transfer RNA (tRNA) accumulation. This tRNA resolves into two isoforms, one of which is gland-specific and quantitatively correlated to its fibroin-synthesizing activity. The second of these waves serves to optimize the gland’s translational milieu through the differential expression of the tRNAs cognate to the most preponderant amino acids of the gland’s fibroin product in a similar proportion to that in which these appear in the fibroin. Worthy of note is the disproportionate accumulation of alanine-tRNA which is produced primarily within the first wave of small RNA syntheses and which selectively enriches the system with a tissue-specific isoacceptor species in a proportion of 4:1 to its constitutive counterpart. The nucleotide sequence of this isoform endows it with structural features which foster its possible performance in other than elongation functions during the synthesis of fibroin.
The flagelliform or coronata glands of the orb-web spider, Nephila clavipes, have been studied and compared to other silk-producing glands from the organism. The glands, which produce silk for the double filament of the core thread in the sticky spiral, exhibit three distinct morphological areas: tail, sac, and duct. Electrophoretic separation of the solubilized contents of the glands yields an uppermost diffuse band of high molecular size, preceded by a stepladder of well-defined peptides, which have been shown to be products of discontinuous translation in three other sets of glands. The luminal contents do not migrate as a discrete and well-defined band as those of the other glands, but rather as a diffuse area, typical of glycosylated proteins. Fibroin synthesis is stimulated by the mechanical depletion of the organism's stored silks, as in other Nephila glands, judged by the increased intensity of the bands and also by the structural alterations seen in cross sections of the glands' tails.
Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.
Der Spinnapparat der tropischen Radnetzspinne Nephila setzt sich aus 6 verschiedenen Drüsensorten zusammen, von denen eine als „Glandula coronata“ erstmals unterschieden wird. Die Verteilung der verschiedenen Drüsen auf die 3 Warzenpaare und der Bau der Spulen werden beschrieben. Bezüglich der Leistung des Spinnapparates ergibt sich als neu vor allem die Bildungsweise des Klebfadens der Netze. Er entstammt einem Aggregat von 3 Spulen, von denen eine den Grundfaden aus dem Sekret der Glandula coronata liefert, während aus den beiden anderen der Klebstoff ausfließt, der sich über jenen ergießt. Der Klebstoff ist, wie bereits bekannt, ein Produkt der Glandulae aggregatae.
Experiments presented here suggest that control of gene expression mediated by juvenile hormone is at the translational level
and involves the appearance of a new transfer RNA and its activating enzyme. The evidence is as follows. Topical application
of juvenile hormone to 1st day pupae of Tenebrio molitor caused a second pupal molt after 7 days, instead of an adult molt. Ribosomes isolated from pupae on the 1st or 7th (last)
day after the larval pupal molt contained messenger RNA for cuticular protein. tRNA and enzyme from hormone-treated animals
caused a 10-fold decrease in the ratio of tyrosine to leucine incorporation on ribosomes from both hormone-treated and control
animals. Co-chromatography of the tryptic digest of 14C-labeled product synthesized on ribosomes in the presence of tRNA from hormone-treated animals with 3H digest of product synthesized with tRNA from normal 7th day pupae showed different elution profiles for the two isotopes.
On purified ribosomes neither chain initiation nor chain termination were observed. The translation of cuticular protein in vitro is affected by the relative amount of tRNA provided from either normal or hormone-treated animals.
We have cloned and characterized a complete set of seven U1-related sequences from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously
studied: one U1 gene at 21D which encodes the prototype U1 sequence (U1a), one U1 gene at 82E which encodes a U1 variant with
a single nucleotide substitution (1Mb), and a pseudogene at 82E. The four previously uncharacterized genes are another U1b
gene at 82E, two additional U1 a genes at 95C, and a U1 gene at 95C which encodes a new variant (U1c) with a distinct single
nucleotide change relative to U1a. Three blocks of 5′ flanking sequence similarity are common to all six full length genes.
Using specific primer extension assays, we have observed that the 1Mb RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-contalning snRNP particles are able to participate in
the process of premRNA splicing. We have also examined the expression throughout Drosophila development of the two U1 variants
relative to the prototype sequence. The U1c variant is undetectable by our methods, while the U1b variant exhibits a primarily
embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse.
Spider major ampullate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed to consist of pseudocrystalline regions of antiparallel beta-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (iii) a 15 amino acid, highly conserved segment. The latter is predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine. The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position. A model for the physical properties of fiber formation, strength, and elasticity, based on this repetitive protein sequence, is presented.
In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.
A small set of distinctive short RNA molecules are found in the nuclei of all higher eukaryotic cells and yeast, in protein complexes known as 'small nuclear ribonucleoprotein particles', or snRNPs. Recent work has confirmed early suggestions that these particles form part of the machinery by which primary RNA transcripts are processed to their mature, functional form. In particular, snRNPs have been shown to be an integral part of the 'spliceosome', a multi-component complex involved in the removal of intron sequences from the coding regions of messenger RNA precursors.
The mechanism by which the scanning ribosome recognizes the first AUG codon nearest the 5' end of eukaryotic messenger RNA
has not been established. To investigate this an anticodon change (3'-UCC-5') was introduced into one of the four methionine
initiator (tRNAi(met) genes of Saccharomyces cerevisiae. The ability of the mutant transfer RNA to restore growth properties
to his4 initiator codon mutant yeast strains in the absence of histidine was then assayed. Only the complementary codon, AGG,
at the his4 initiator region supported His+ growth. The mutant transfer RNA also directed the ribosome to initiate at an AGG
placed in the upstream region of the his4 message. Initiation at this upstream AGG precluded initiation at a downstream AGG
in accordance with the "scanning" model. Therefore, an anticodon: codon interaction between tRNAi(met) as part of the scanning
ribosome and the first AUG must function in directing the ribosome to the eukaryotic initiator region.
The production of silk is a specialized case of the capacity of organisms to produce fibrous proteins. Silks are produced for a variety of life needs, and their production is widespread within the animal kingdom, particularly in the phylum Arthropoda, and more specifically in the classes Insecta and Arachnida. Of the insects, the best-known silk producers are the moths and butterflies, and within the arachnids, the spiders. The best-characterized of the fibroin synthesizers has been the silkworm, Bombyx mori. The system is known for its massive production of silk, late in the last molt or fifth instar.
All eukaryotic cells contain multiple small nuclear RNAs, designated U-sn At present ten U-snRNAs have been identified; they account for about 1% total mammalian cellular RNA. Of these, the U1 to U8 snRNAs have been sively characterized. All the U-snRNAs contain a 5′-cap structure consis a blocked 5′-terminal pyrophosphate linkage; except for U6 RNA, a tri guanosine-containing “cap” is the 5′-end. These U-snRNAs are confined nucleus and are metabolieally stable. They are synthesized by RNA polyII. U6 snRNA, which has a non-nucleotide cap structure, is synthesized by RNA polymerase III. All the U-snRNAs are present in the cells as small ribonucleopro- tein particles (snRNPs), that are metabolically stable.
The location and function of the five or six sets of silk glands of Araneus diadematus (Cl) are discussed. The structure and function of the three major parts of the ampullate gland indicate a synthesizing, collecting, and possibly structuring section. Two methods of stimulation of the ampullate gland, namely emptying the gland and cholinergic stimulation, are known. In both cases there is an initial secretory stage followed by rapid synthesis of new protein. The sequence of events following stimulation by both methods is described, based on studies of the incorporation of labeled protein and RNA precursors and on autoradiographic studies. Characteristic changes occur in the fine structure during the stimulatory cycle. Several experiments show that the spider has information on the amount of silk available to it for use in web-building. A structure which may act as a biological transducer has been located in the ampullate gland.
The major class of small nuclear ribonucleoproteins (snRNPs) are part of the nuclear machinery for processing pre-messenger RNAs. The genes encoding the RNA components of these particles (snRNAs) have several unique features which distinguish them from other pol II- and pol III-transcribed genes. Assembly of the snRNAs with proteins to form snRNP particles has been studied in vivo, and new in vitro systems promise to advance our understanding of the assembly process still further.
Die t-RNS Arten des Spinndruse der Seidenraupe (Bombyx mori L) wurden an verschiedenen Tagen des letzten Larvenstadiums aus dem hinteren sezernierenden Anteil der Drüse (Secreteur) und aus dem mittleren Teil (Reservoir) extrahiert und mittels Gegenstromverteilung in einem speziellen System von Salzlösungen und organischen Lösungsmitteln aufgetrennt. Sowohl die Entwicklung der Absorptions Profile der t-RNS von der Phase des Gewebswachstums bis zur Phase optimaler Fibroin und Sericinsekretion als auch die Charakterisierung und Bestimmung durch heterologe Acylierung der 11–17 t-RNS haben gezeigt, dass eine Anpassung der t-RNS-Arten an den Transport der jeweilig vorherrschenden Aminosäuren erfolgt: Gly, Ala, Ser, und Tyr im Falle des Fibroins im «Secreteur und Ser, Gly, Asp, und Ala im Falle des Sericins im «Reservoir.
Die t-RNS Arten des hinteren, sezernierenden Teills des Spinndrüse (Secreteur) der Seidenraupe (Bombyx mori L) und des mittleren Teils (Reservoir) sind während der Wachstumsperiode und während der Sekretion von Fibroin und Sericin im letzten Larvenstadium aus dem postribosomalen Uberstand extrahiert und ihre quantitative Entwicklung verfolgt worden. Im sezernierenden, Fibroin produzierenden Drüsenteil nimmt die t-RNS vom ersten bis achten Tag um einen Faktor 10 zu, um in den letzten beiden Tagen vor dem Spinnen des Kokons rasch wieder abzunehmen; im Reservoir-Anteil, der Sericin produziert und den Seidenfaden stapelt, läuft die Entwicklung parallel: vom ersten bis siebten Tag um einen Faktor 5.
Die individuelle Entwicklung der 17 t-RNS Arten ist mittels heterologer Acylierung in vitro untersucht worden, nachdem die optimalen Bedingungen dafür ausfindig gemacht worden waren. Wir haben gefunden, dass die t-RNS Arten vorherschen, die den häufigsten Aminosäuren der Seidenproteine entsprechen: die t-RNSAla, t-RNSGly, t-RNSSer und t-RNSTyr im Secreteur und die t-RNSSer, t-RNSAla, t-RNSGly, t-RNSGlu, t-RNSAsp im Reservoir machen am 8. bzw. am 7. Tag 2/3 des ganzen t-RNS aus.
Eine unterschiedliche Entwicklung zweier t-RNS Gruppen wurde festgestellt: auf der einen Seite die t-RNS Arten, die an der Herstellung von Fibroin und Sericin mitwirken, mit einer maximalen Konzentration am 7. und 8. Tag, auf der anderen Seite die t-RNS Arten, die an der Synthese der übrigenProteine beteiligt sind, mit einem Maximum am 4. bis 5 Tag.
1.1. The amino acid composition is reported for the secretions present in the various silk glands in the female garden spider, Araneus diadematus.2.2. The presence of the flagelliform gland is confirmed, and it is shown that the large and the small ampullate glands produce different types of secretion. The number of different silk glands in an adult female amounts thus to seven.3.3. The composition of the secretions is discussed in relation to the known and assumed functions of the glands as well as in relation to the mechanical properties of the different types of silk produced by spiders.
The tissue-specific fibroin product of the large ampullate glands of the spider Nephila clavipes was isolated and analyzed with respect to homogeneity and size. The solubilized luminar contents of the glands migrate as a homogeneous band in SDS electrophoresis. Its molecular mass has been estimated to be approximately 323,600 daltons. Excised glands have been manintained metabolically active under rather simple culture conditions. Protein synthesis, monitored by incorporation of labeled alanine and glycine, has been elicited through exposure to a cholinergic agent in the culture medium as well as by depleting the animals of their store of silks.
1.1. Synthesis of fibroin has been elicited in the cultured large ampullate glands of the spider Nephila clavipes.2.2. A dramatic wave of protein synthesis ensues 60 min after stimulation, preceded by a wave of RNA synthesis.3.3. The glands in culture synthesize the full-size protein product.4.4. Both stimulus-elicited responses are expressed exclusively by the gland tissue.5.5. In addition to the luminar product, the secretory epithelium contains an array of lower molecular size peptides bearing a striking resemblance to those reported as products of translational pauses during the synthesis of silk in Bombyx mori.
Conditions have been determined under which reverse transcriptase catalyzes the synthesis of high yields of full length complementary
deoxyribonucleic acid (cDNA). These conditions depend not only on the concentration of deoxynucleoside triphosphates (1) but
also on the concentration of reverse transcriptase. An analysis of the kinetics of cDNA synthesis and the size of cDNA synthesized
as a function of time under different conditions indicates that the mechanism of action of reverse transcriptase is partially
distributive. This accounts for the neccessity of a high enzyme concentration to obtain high yields of full length cDNA. Additional
experiments indicate that the yield of cDNA is limited by the fact that the template mRNA is rapidly inactivated. This is
most likely due to the fact that the product cDNA is hydrogen bonded to the template mRNA during synthesis.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
In the silk gland of the silkworm Bombyx mori L. the iso-tRNA population is quantitatively adjusted to codon frequency of fibroin mRNA. Such a quantitative correlation has been found in the Rabbit reticulocyte. So far the quantitative adaptation of iso-tRNA species to codon frequency of mRNA being translated appears to be a general phenomenon, the mechanism of which — constitutive or adaptative — remains obscure.The comparison of sequenced mRNA shows that codon frequencies differ significantly between viral and animal mRNA. In addition, messengers of animal tissues have common features of a family: near absence of CG doublet ending codons, low frequency of A ending codons, high frequency of C ending codons. We found a significant correlation between synonymous codon frequencies in animals and the distribution of corresponding isoaccepting tRNA species estimated from different animal tisues. Our analysis deals with the prominent four amino acids of proteins (alanine, glycine, leucine and serine), which correspond to twenty codons and thirteen tRNA species. On the contrary, the animal iso-tRNA distribution is not correlated with viral mRNA codon frequency or possibly some plant mRNA (at least for tRNALeu).The agreement between the non random distributions of codons and iso-tRNA species in animals could probably indicate that codons and anticodons have been simultaneously selected during evolution to optimize translation accuracy.
The nucleotide sequences of two major alanine tRNAs from the Bombyx mori posterior silk gland have been determined. One of these tRNAs appears to be specific to the silk gland, where its accumulation is associated with the rapid production of fibroin. Both sequences are identical, with the exception of a single nucleotide in the anticodon stem. A striking feature of both alanine tRNAs is that loop IV contains sequences previously believed to be restricted to initiator tRNA.
Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis, oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis. We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo, namely, increased metabolic stability.
Sequences for 5S RNA from somatic cells and oocytes of Xenopus mulleri are presented. Comparison with sequences previously given for Xenopus laevis indicates that the somatic 5S RNA genes of each species are more closely related to each other than either is to its own set of oocyte genes, suggesting that somatic and oocyte genes within each species are evolving independently. However, detailed analysis of sequence variants in each species suggests that there is a mechanism which allows occasional genetic exchanges between somatic and oocyte-specific genes. Possible genetic mechanisms which allows such an exchange are discussed.
In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA(Glu) is common to the biosyntheses of both porphyrins and proteins.
Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA(Lys), tRNA(Ile), tRNA(Thr), and tRNA(Tyr), which comprise a structurally related family, tRNA(Lys) is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. The Galago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.
Electron microscopic studies of unstimulated and stimulated spider fibroin glands show that the fibroin synthesis stimulus evokes visible changes in both the endoplasmic reticulum and Golgi apparatus of their secretory epithelium. Gradual increase in distension of the reticulum accompanies the increase of evoked fibroin synthetic activity. The flattened translucent Golgi vesicles, seen in inactive cells, display a gradual increase in size and number, also with time. The stimulation also elicits a gradual transition in the gland's luminal membrane, during which the microvilli on the lining gradually disappear acquiring an electron dense appearance. Correlations of the observed transitions to the gland's increase in rate of elicited synthetic activity are discussed. The parallelisms between the ultrastructural modifications observed in the spider secretory cells with those described in the silkworm glands during their progression through the fifth instar have been stressed.
The large ampullate glands of the orb-web spider, Nephila clavipes provide massive amounts of fibroin throughout the lifetime of the adult female. We have developed methods to culture the glands and manipulate their biosynthetic activity. This has allowed us to monitor a series of molecular events that precede silk production in glands excised from appropriately stimulated animals. In this paper, we demonstrate that prior to the transient dramatic production of fibroin, such glands accumulate large amounts of tRNAs cognate to the abundant amino acids in spider silk. One of these, alanine tRNA, appears to consist of two isoaccepting forms--one constitutive, and the other silkgland specific. Moreover, the silkgland-specific form appears to accumulate preferentially in response to stimulation. This phenomenon of tissue-specific tRNA production appears similar to that found in the silkglands of Bombyx mori, but the spider system has the unique property of permitting manipulation in vitro. Thus, it provides an unusual opportunity to study the mechanism of regulated tRNA synthesis.
During the course of a cloning project many occasions occur when it is necessary to concentrate nucleic acid samples or change the solvent in which a nucleic acid is dissolved. Fulfillment of these requirements is met by nucleic acid precipitation techniques. Described are ethanol and other precipitation techniques.
The majority of the genes for U1 RNA are organized in tandemly repeated units in the sea urchin. To assess the level of expression of these genes in the sea urchin Lytechinus variegatus, we measured the transcription of sequences 3' to the gene. The tandemly repeated U1 genes are expressed in morula and continue to be expressed at high rates until 2 hr after hatching, at which time the rate of expression of all the U1 genes and the tandemly repeated U1 genes declines sharply. By the gastrula stage the synthesis of total U1 RNA has declined by a factor of 8. The major tandemly repeated genes are inactive by this time, although other U1 genes remain active. The sequence of U1 RNA synthesized late in embryonic development differs from the sequence of U1 RNA encoded by the tandemly repeated set of U1 RNA genes, indicating that there must be other U1 RNA genes that are active late in embryonic development.
Optimum efficiency of protein biosynthesis is obtained through continuous, selective adjustment of cytoplasmic tRNA population to the amino acid composition of the proteins being synthetized. We call this adjustment “functional or quantitative adaptation”. This regulation phenomenon has been found in highly differentiated cell systems such as the silkgland of Bombyx mori L., reticulocytes, plasmocytoma and lens. It can be applied to other cell systems such as Escherichia coli, yeast or liver. Implications of functional adaptation of tRNA are discussed in terms of a modulated tRNA biosynthesis with each tRNA species regulated independently.
1.1. A single injfected dose of acetylcholine into the spider, Araneus sericatus, causes secretion of performed fibroin from the ampullate silk glands. This is followed by increased protein synthesis, measured by incorporation of labelled amino acids and nucleic acid precursors.2.2. Puromycin inhibits the increased synthesis of new protein but does not affect the initial secretion. Thus the processes of secretion and synthesis can be separated.3.3. A scheme is proposed for the overall regulation—both central and local— of the protein production in the ampullate gland of Araneus sericatus.
The transcription of 5 S RNA genes during oogenesis results in the storage of sufficient 5 S RNA in ribosomes to support subsequent embryogenesis. Xenopus oocytes of all stages synthesize oocyte-type 5 S RNA. A generalized repression of transcription occurs at meiosis and is maintained throughout early cleavage. The onset of 5 S RNA synthesis is detected at approximately the 4000-cell blastula stage (stage 9), concomitant with de novo synthesis of other species of RNA. At this developmental stage the level of 5 S RNA synthesis is low relative to the synthesis of tRNA and small nuclear RNAs. Analysis of this newly synthesized 5 S RNA reveals it to be a nearly equal mixture of oocyte and somatic 5 S RNA derived from both maternal and paternal genes. Given the 50:1 ratio of oocyte to somatic 5 S RNA genes in X. laevis, these results indicate that the majority of the oocyte 5 S RNA genes are inactivated at this time. This reflects differential transcription of the two families of 5 S RNA genes rather than post-transcriptional stability as demonstrated by the ability of a chromatin template isolated from stage 9 embryos to direct the same ratio of oocyte to somatic 5 S RNA synthesis in vitro as that observed in vivo. By completion of gastrulation, 5 S RNA synthesized in vivo and directed from chromatin in vitro is at least 90% somatic 5 S RNA. These results are consistent with a model in which the decrease in concentration of the 5 S-specific transcription factor relative to the number of 5 S RNA genes during embryogenesis contributes to the inactivation of the oocyte 5 S RNA genes.
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There has been considerable speculation about what features in messenger RNA direct ribosomes to the site where peptide-bond formation is to begin. In prokaryotic systems, a substantial body of evidence supports Shine and Dalgarno’s proposal (1974) that base pairing occurs between the pyrimidine-rich 3’-end of 16S ribosomal RNA and a purine-rich sequence located approximately ten nucleotides to the left of the AUG initiator codon (Steitz and Jakes 1975; J.J. Dunn et al. 1978; Steitz 1979). This interaction plays a central role in recognition of initiation sites by bacterial ribosomes, although other features in prokaryotic mRNAs also influence the efficiency of ribosome binding (Lodish 1970; Taniguchi and Weissmann 1978; Borisova et al. 1979; Fill et al. 1980; Iserentant and Fiers 1980). The structural similarities between prokaryotic and eukaiyotic ribosomes (Boublik and Hellmann 1978; Gourse and Gerbi 1980; Tanaka et al. 1980) and the obvious parallels in the overall process of peptide-bond formation indicate a high degree of conservation during evolution of the protein synthesis machinery.
Neither a dynamic nor an energetic approach of the translation process has taken into account that intracellular levels of iso-tRNA species are adapted or adjusted to the codon frequency of mRNA being decoded (Bombyx mori silk gland, rabbit reticulocyte). A critical study of available experimental data suggests that the average elongation rate of a protein is maximized in the presence of an adapted tRNA population, usually an homologous tRNA. In addition, the amount of synthesized protein parallels that of corresponding mRNA. Other evidences--including in vitro and in vivo elongation assays with fibroin mRNA--show that individual elongation rates are not uniform. Pauses occur at certain sites of the mRNA chain. The relative lifetime of these pauses depends on the tRNA pool used. Finally, it appears that translation accuracy also depends on the balanced tRNA population. We propose to explain these different effects by using a codon-anticodon recognition model, called "trial and error system" based on a stochastic processing of the ribosome. Accordingly, various acylated tRNA species which surround a ribosome randomly encounter the receptor A site. Every trapped tRNA species is tested for a proper pairing with the codon to be recognized at the level of a comparator or discriminator function. If the pairing is correct, transpeptidation becomes irreversible. If not, the aminoacyl-tRNA is rejected and another randomly trapped tRNA is processed in turn. Mathematical analysis of this model shows that the mean number of trials used for translating the whole sequence of a mRNA is minimized when the proportion of different iso-tRNA species is correlated with the square root of codon frequency. Quantitations of reticulocyte tRNA support such a parabolic relation. Our translation system model brings some light into the role of tRNA adaptation for optimizing translation efficiency, i.e. maximizing both speed and accuracy. Some consequences of the model are discussed.
The amino acid composition of 74 fibroins from various silk-producing arthropods has been determined, 26 of these fibroins being analysed quantitatively, and the remainder semi-quantitatively after paper chromatography. The results show that there are wide variations in the amino acid compositions of the fibroins, although all those examined so far are characterized by the high proportions of the simple amino acids, glycine, alanine, and serine, that they contain. No general correlation could be found between the biological classification of the arthropods and the amino acid composition of the fibroins they produce, though a few limited correlations were observed. In particular, the fibroins produced by the Saturniidae resemble one another in amino acid content and always contain more alanine than glycine. Some fibroins from biologically unrelated species have closely similar amino acid compositions and crystal structures. The significance of the results in relation to the crystal structure, taxonomy, and biological function of the proteins is discussed.
tRNA adaptation during fibroin synthesis in thecultured glands of
- R Dompenciel
The two alanine transfer RNA in
- C E Carrasco
tRNA-tRNA interaction alter the rate oftranslation
- D Smith
Evaluation of the scanning model for theinitiation of protein synthesis in eukaryotes
- M Kozak
Biochemical research on oogenesis. Oocytes and liver cells ofthe teleost fish
- H Dennis