Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

Reiteration frequency of procollagen genes in the guinea pig genome: Collagen genes are not amplified during granuloma fibroblasts differentiation

Procollagen mRNA was purified from collagen synthesizing polysomes obtained from an experimental guinea pig granuloma, and iodinated in vitro. The procollagen ¹²⁵I-labelled mRNA was hibridized with granuloma and liver guinea pig DNA in vast DNA excess conditions. A Cot $\text{1}\text{2}$ 800–900 mol · s · l^?1 for both tissues was obtained from the hybridization curves. With these results, we could suggest the existence of 11–13 procollagen genes per haploid genome. By the analysis of the hybridization data it was possible to infer that there is no genomic amplification in tissues highly specialized in the synthesis of collagen such as granuloma.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

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.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

A procedure for the cloning and identification of Y-specific middle repetitive sequences in Drosophila hydei

Clones of hybrid plasmids containing moderately and highly repeated sequences of Drosophila hydei exhibit positive autoradiographic signals if hybridized to labeled whole genome DNA. Such clones were screened with labeled male ($\text{X}\text{Y}$) and female ($\text{X}\text{X}$) DNA, and male-specific fragments were identified. Further hybridization of male-specific clones to female $\text{XX}\text{Y}$, and male $\text{X}\text{0}$ DNAs established them as containing Y-specific moderately repeated sequences. Further verification of one particular cloned fragment as Y-specific is presented and possible applications of this procedure are briefly discussed.     

The fine structure of silk fibroin

The fine structure of Bombyx mori silk fibroin was investigated by electron microscopy and X-ray diffraction techniques. Examination of silk fibers fragmented with ultrasonic radiation and negatively stained revealed the presence of ribbon-like filaments of well-defined lateral dimensions. Analysis of the breadths of the equatorial reflections in the X-ray diffraction pattern of fibroin yielded similar dimensions for the lateral extent of the crystallites. It is concluded that the crystalline material in B. mori silk fibroin is in the form of ribbon-like filaments of considerable length parallel to the fiber axis and of lateral dimensions approximately 20 x 60 A.     

Studies on Agrobacterium tumefaciens. IV. Nonreplication of the bacterial DNA in mung beam (Phaseolus aureus).

DNA·DNA filter hybridization and DNA solution enrichment reassociation experiments showed that no $\text{Agrobacterium tumefaciens}$ DNA was replicated in mung bean seedlings under the conditions specified in published reports for the uptake, integration, and replication of bacterial DNA in higher plants. Residual presumptive DNA hybrids that formed in a few instances were characterized by thermal chromatography on hydroxylapatite. The Tm and melting profiles of these hybrids from DNA-treated plants were the same as those from untreated control plants. The sensitivities of these procedures were sufficient to detect $\text{A. tumefaciens}$ DNA in the order of 0.005% to 0.01% of the plant genome. These results do not concur with previous reports that large pieces of DNA (at least 30%) of the plant genome of bacterial-DNA-treated-plants is made up of bacterial donor DNA.     

The DNA content of sperm and hemocyte nuclei of the silkworm,Bombyx mori L.

To estimate the size of the haploid genome of the silkworm, Bombyx mori (Lepidoptera), amounts of Feulgen-DNA staining in individual nuclei of primary spermatocytes, spermatids, maturing sperm, and larval or pupal hemocytes were determined with an integrating microdensitometer and compared with the Feulgen-DNA levels found for chicken erythrocyte nuclei, or the sperm and erythrocyte nuclei of Xenopus laevis that were included with each Bombyx preparation as empirical reference standards of 2.5, 3.15, and 6.3×10^?12 g DNA per cell, respectively. Under these conditions, the haploid male genome of B. mori was estimated as 0.52±0.01 (S.E.)×10^?12 g DNA, corresponding to a molecular weight of roughly 3.1×10¹¹ daltons. From similar measurements of Feulgen-stained hemocyte nuclei, approximately 1.0±0.05 (S.E.)×10^?12 g DNA was estimated for the diploid or 2C male genome of Bombyx. These values compare favorably with estimates of genome size based upon analysis of the kinetics of reassociation of DNA isolated from B. mori and provide an independent basis for assessing the degree of polyploidy achieved by the giant nuclei in the posterior silk gland prior to its secretion of fibroin at the end of the fifth larval instar.     

The genes for silk fibroin in Bombyx mori

The genes for the protein silk fibroin were quantitated by hybridizaton of purified fibroin messenger RNA with the DNA from several tissues of the silk-worm Bombyx mori.     

Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses.

The genome of the genetically transmitted endogenous C type virus of chickens, RAV-O, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of ¹²⁵I-labeled genomic RNA demonstrated that the genome of RAV-O lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (td RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-O sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3′ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-O were concentrated within 40% of the sarcoma virus genome from the 3′ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-O 3′ end fragments demonstrated that the genomic sequences of RAV-O missing in RSV were concentrated at the 3′ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome.     

Pulse-label analysis and mapping of the two terminal regions of asynchronous complementary strand replication of mitochondrial DNA in transformed hamster cells

In vivo pulse-chase radioactive labeling studies were performed to localize within the physical map of $\text{C}{}_{13}\text{B}{}_4$ hamster mtDNA² the two terminal regions of heavy and light complementary strand synthesis. These terminal segments have been defined operationally as that region on the H- and L-strand that is synthesized last. mtDNA of monolayer cultures was pulsed with [³H]thymidine for a minimum period of 10 minutes, which is about one-tenth of one round of mtDNA synthesis, followed by chase periods of up to 120 minutes. The properties of the labeled closed circular replicative intermediates E-mtDNA, C-mtDNA and D-mtDNA were analyzed in $\text{CsCl}\text{PrI}{}_2$ gradients and in neutral sucrose velocity and alkaline CsCl gradients. Both terminally labeled α and β daughter molecules were found to pass through the E-mtDNA stage. Sensitivity of $\text{C}{}_{13}\text{B}{}_4$ mtDNA to alkali and ribonuclease A indicated the presence of covalently linked ribonucleotides. The distribution (specific activity) of pulse-chase radioactivity relative to uniform label was followed in electrophoretically separated HpaII + HinIII and HpaI restriction fragments (freed of 7 S initiation sequences) and corrected for thymine content. The strand specificity of the pulse-label was determined by hybridization of restriction fragments with H- and L-strands of mtDNA. The kinetic data agree precisely with electron microscopic determinations of H- and L-strand origins at respective genome positions of 0 and 67 ± 3, which are located on HpaII + HindIII fragments 9 and 6, respectively (Nass, 1980). The two terminal regions are within the predicted genome sector between about 67 and 100/0 map units; the highest terminal pulse-chase radioactivity extends within 5 to 15% of the genome's length behind each origin. The kinetics of early labeling events were found to differ at the two termini. The evidence indicates that the majority of L-strand initiation/termination sites are in the region near map position 67 ± 3, and confirms the highly asynchronous replication mechanism of this DNA.