Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori.

The number of silk fibroin genes per genome in the silkworm Bombyx mori has been determined by hybridization using fibroin [¹²⁵I]mRNA. The purified [¹²⁵I]mRNA had an oligonucleotide pattern after RNAase T₁ digestion which was characteristic of fibroin mRNA (Suzuki &; Brown, 1972) and it hybridized specifically to DNA with a G + C content expected for a fibroin gene. Thermal denaturations indicated that these hybrids were mismatched by about 3%, which probably indicates some variation among the sequences encoding the internal repetitions of the fibroin protein.The concentration of fibroin gene sequences in B. mori DNA was measured by saturation hybridization of [¹²⁵I]mRNA to filter bound DNA. The same saturation level of 1.8 × 10^?5 μg mRNA per μg DNA was calculated from data obtained with unfractionated DNA and with fibroin gene sequences which had been separated from bulk B. mori DNA by actinomycin $\text{D}\text{CsCl}$ centrifugation. Scatchard plots of the subsaturation data extrapolated to an identical saturation value. Internal reiteration of the fibroin mRNA molecule was apparent from the high association constant of hybridization. An exhaustive hybridization experiment showed that such repetitions comprise at least 90% of each mRNA molecule. The saturation value, in conjunction with the genome DNA content and the mRNA size, indicated the presence of only one fibroin gene per haploid B. mori genome.Hybridization of actinomycin $\text{D}\text{CsCl}$ fractionated DNA indicated that fibroin mRNA can form hybrids with DNA that bands with bulk B. mori DNA. These hybrids appear to involve DNA which is related to, but distinguishable from, true fibroin gene sequences. The fibroin gene-related sequences form mismatched hybrids with the mRNA, are much shorter than the fibroin gene and are dispersed in B. mori DNA of much lower G + C content, and there are many copies of these sequences per B. mori genome.     

小麦族植物DNA重复序列研究

近些年来从小麦族植物中分离到了许多DNA重复序列,并对其组织结构特点,物种分布特异性和在染色体上的分布特征做了广泛的研究,其中一些重复序列已被成功地用于检测遐入小麦的外源染色质和小麦族有关种属的进化研究,本文就以上诸方面进行了简要介绍。     

Repeated DNA sequences in fungi

Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10-20% repeated DNA sequences. There are approximately 100-110 copies of repeated DNA sequences of approximately 4 × 10⁷ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5°C difference of T_e50 (temperature at which 50% duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA.     

Repeated DNA sequences in mycobacteria

In tuberculosis, it is often important to establish the source of infection and to determine whether disease is due to a new strain of Mycobacterium tuberculosis or to relapse. To cope with the resurgence of tuberculosis and atypical mycobacterioses in AIDS patients, on the one hand, and to overcome the limitations of classical bacteriological procedures on the other, the development of rapid, sensitive, and reliable diagnostic and epidemiologic tools is highly desirable. Molecular typing methods are often based on repeated genes such as those for rRNA. Ribotyping is of limited use with pathogenic mycobacteria. as the slow-growers possess a single rRNA operon, while the fast-growers have two. This problem has been overcome by the discovery and study of repeated DNA elements in mycobacterial genomes, as these provide an alternative pathway for diagnostic and epidemiological investigations.     

Sequence polymorphisms around the 5'-end of the silkworm fibroin H-chain gene suggesting the occurrence of crossing-over between heteromorphic alleles

Nucleotide sequences around the 5'-ends of the silkworm fibroin H-chain genes of the three strains, Nd(2), J-139, and F1(Gunka X Hoshun), of Bombyx mori were determined. Comparison of the sequences among these strains and the sequences reported previously for the two other strains, F1(Gunpo X Shugyoku) and Daizo, indicates that polymorphisms are present in the 5'-flanking and intron regions and that each region has at least two sequence variants independent of each other. These results suggest that crossing over between the heteromorphic H-chain alleles has occurred during the breeding of these strains.     

Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences

The DNA sequence orgainzation of the protein encoding region of the gene for silk fibroin has been analyzed. The accompanying paper (Manningm R. F., and Gage, L. P. (1980) J. Biol. Chem. 255, 9451-9457) shows that the total length of the gene, and its protein, as well as the pattern of restriction sites in the gene is highly polymorphic among inbred stocks of Bombyx mori, In this paper, those features of fibroin gene structure which are invariant among these alleles are presented. Fibroin is composed primarily of relatively short "crystalline" and "amorphous" peptides of known sequence whose arrangement in the protein is unknown. Knowledge of the codons most commonly used in fibroin mRNA allowed utilization of particular restriction inzymes as a means for determing the nature and organization of crystalline and amorphous coding sequences in the fibroin gene. Three restriction endonucleases were identified that cleve sequences coding for amorphous region peptides. Their cleavage pattern revelaed that the repetitive coding sequence of the gene core (approximately 15 kilobases) is divided into at least 10 large crystalline coding domains interrupted by smaller amorphous coding domains. Many restriction endoncleases do not cleave the fibroin core at all, three of them with four gase recognition sequences. Specific deductions as to codon usage and repetitive sequence homogeneity in the gene follow from these results. One novel finding is the rigorous exclusion of the glycine codon GGA prior to serine codons even though this glycine codon is used frequently prior to alanine codons. The sequence homogeneity and the regularly alternating arrangement of crystalline and amorphous coding sequences of the gene are discussed in terms of the function of fibroin protein and the evolution of highly repetitive DNA.     

Homeodomain binding sites in the 5' flanking region of the Bombyx mori silk fibroin light-chain gene

Multiple A + T-rich stretches in the 5' flanking region of the Bombyx mori fibroin light-chain gene have been shown to bind two Drosophila homeodomain proteins, EVE (even-skipped) and ZEN (zerknüllt), with high affinities. Some of these sites fall into a class that has the established consensus sequence of the binding sites (TCAATTAAAT) for a diverse group of Drosophila homeodomain proteins, while others are quite heterogenous except that they all possess a core TAAT motif. Since clusters of homeodomain binding sites can also be found in the promoters of other silk protein genes, the fibroin gene and the sericin-1 gene, these observations suggest a possible involvement of some homeobox genes in the regulation of a group of silk protein genes.     

Repeated sequences in the DNA of the eukaryotic genome

Long DNA molecules from a cucumber satellite, the cucumber main band, mung bean, and Chinese hamster ovary (CHO) were digested with mung bean nuclease I, which was used as a probe for high AT regions. The digests were viewed under the electron microscope, and the distribution of sizes for the fragments of nuclease-treated plant DNA showed that the main band cucumber and the mung bean have regions along their genomes spaced at approximately 0.3 to 0.4 μ that are sensitive to the nuclease. The satellite from the cucumber contains these sites at intervals generally of 0.1 μ or less, whereas CHO DNA has these regions at intervals of 0.05 to 1.40 μ in length. The long DNA from the main band of the cucumber and the CHO were also partially melted in formamide at 37°C to denature preferentially the regions along the DNA molecules that are rich in AT. Measurements of the distances from the center of each loop to the center of the adjacent loops showed that these distances for the main band cucumber DNA tended to occur at approximately every 0.4 μ, whereas the corresponding distances for the Chinese hamster DNA were less regular, occurring every 0.1 to 1.0 μ.     

Repeated sequences on mitochondrial DNA ofSpirodela oligorhiza

Summary Mitochondrial DNA ofSpirodela oligorhiza (duck weed) was analyzed with restriction enzymes. The genome size appears to be at least 250 kbp. Four different PstI fragments were cloned. These four clones contain a sequence which is reiterated about 100-fold on theSpirodela mitochondrial DNA. Hybridization analysis showed that a similar sequence is present onZea mays mitochondrial DNA, although much less reiterated here. The presence of these reiterated sequences might contribute to the physical heterogeneity of plant mitochondrial DNA.