Short Inverted Repeat Sequences Research Articles

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation.

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats

DNA sequences that read the same from 5′ to 3′ in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as ‘start codons’). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

Abstract 2732: Short inverted repeats are hot spots for genetic instability in mammalian cells

Abstract Analyses of chromosomal aberrations in genetic disorders such as leukemia and lymphoma have provided compelling evidence that segments of the human genome are prone to breakage that lead to DNA rearrangements. Many of these hot spot areas contain sequences that have the potential to form non-B DNA such as Z-DNA, H-DNA or cruciforms. Although research has shown that large palindromes are mutagenic in mammalian cells, the instability of short inverted repeat sequences (30 bp), which are mapped near breakpoints and quite abundant in the eukaryotic genome, has not been investigated. In order to study the effects of short cruciform-forming sequences on genetic instability, we subcloned short AT- and CG-rich inverted repeats into a lacZ’ mutation-reporter shuttle vector. The cruciform structures that formed at the palindromic sequences were probed using S1 nuclease to confirm the presence of the non-B DNA structure. Cruciform-induced mutagenesis was measured in mammalian COS-7 cells via transfection then blue-white screening in DH5α cells. Ligation-mediated PCR provided direct evidence that cruciform-forming sequences induced double-stranded breaks in mammalian cells. In vitro replication assays using SV40- HeLa cell-free extracts were employed to establish the connection between replication and cruciform-induced genetic instability. Results from our study demonstrate for the first time that cruciform structures that form at short inverted repeats are mutagenic in mammalian cells. The majority of the mutants induced by cruciforms were large-scale deletions that spanned the inverted repeat sequence. Analyses of junctions revealed that >80% of the mutants contained microhomologies which are characteristic of error-prone nonhomologous end-joining repair. This suggests that cruciform structure is cleaved by replication-independent, structure-specific nucleases in mammalian cells that result in DNA double-stranded breaks at positions directly adjacent to the inverted repeat. These results indicate that cruciform-forming sequences may play a role in genetic instability and contribute to human diseases associated with gene translocations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2732. doi:10.1158/1538-7445.AM2011-2732

PigS and PigP Regulate Prodigiosin Biosynthesis inSerratiavia Differential Control of Divergent Operons, Which Include Predicted Transporters of Sulfur-Containing Molecules

Serratia sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Regulation of prodigiosin biosynthesis involves a complex hierarchy, with PigP a master transcriptional regulator of multiple genes involved in prodigiosin production. The focus of this study was a member of the PigP regulon, pigS, which encodes an ArsR/SmtB family transcriptional repressor. Mutations in pigS reduced production of prodigiosin by decreasing the transcription of the biosynthetic operon. The pigS gene is the first in a four-gene operon, which also encodes three membrane proteins (pmpABC) of the COG2391 (DUF395; YedE/YeeE) and COG0730 (DUF81; TauE/SafE) families that we propose constitute transport components for sulfur-containing compounds. We provide the first experimental evidence confirming the membrane localization of a COG2391 protein, that of PmpB. Divergently transcribed from pigS-pmpABC is a bicistronic operon (blhA-orfY), which encodes a metallo-β-lactamase and a coenzyme A-disulfide reductase containing a rhodanese homology domain, both of which may participate in reactions with sulfur-containing compounds. The overproduction of the BlhA and OrfY enzymes and the PmpABC membrane proteins differentially affected pigmentation. We have dissected the contributions of these various proteins and determined their importance in the control of prodigiosin production. PigS-mediated control of prodigiosin occurred via binding directly to a short inverted repeat sequence in the intergenic region overlapping the predicted -10 regions of both pigS and blhA promoters and repressing transcription. PigP was required for the activation of these promoters, but only in the absence of PigS-mediated repression.

Discovery of Stable and Variable Differences in the Mycobacterium avium subsp. paratuberculosis Type I, II, and III Genomes by Pan-Genome Microarray Analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.

Interarm Interaction of DNA Cruciform Forming at a Short Inverted Repeat Sequence

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100–200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3′-half of the repeat, thereby forming an unusual structure in which the 3′-half of the inverted repeat, but not the 5′-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3′-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae.

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

Forced Integration of Moloney Murine Leukemia Virus DNA with a Mutant Integration Site Occurs through Recombination with VL30 DNA

The integration of retrovital DNA to form the provirus requires the presence of short inverted repeat sequences at the termini of the linear vital DNA. We have previously described the construction of a mutant of Moloney murine leukemia virus, carrying an 8-bp deletion in the U5 terminus, that is defective in establishing the integrated provirus. We have now used a selectable marker to allow recovery of rare proviruses formed after transduction from NIH/3T3 cells by retroviral vectors carrying this mutation. Analysis of two such proviruses showed that both were recombinants between the vector and VL30 DNA and arose such that the VL30 sequences could provide an intact terminal sequence for integration.

Mutational Analysis of the Sequences at the Termini of the Moloney Murine Leukemia Virus DNA Required for Integration

The insertion of retroviral DNA into the genome of the infected cell to form the integrated provirus requires the presence of short inverted repeat (IR) sequences at the viral DNA termini. To examine the sequence requirements at the IRa for integration of the Moloney murine leukemia virus, we generated a series of mutants with deletion and substitution mutations and tested their ability to replicate and form proviruses in vivo. The experiments did not detect evidence of a requirement for sequences outside the IRs, and only a very loose requirement for the IR sequences, with many point mutations well tolerated. Examination of the mutants suggests that bases very near the terminus and those approximately one turn of the DNA helix away from the terminus are important for recognition by the integrase function.

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7-9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed.

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage T7 DNA packaging: III. A “Hairpin” end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

Molecular basis of spontaneous mutation at the aprt locus of hamster cells

Mutations occurring spontaneously at the hamster aprt locus were examined at the base-pair level by amplifying target sequences using the polymerase chain reaction and then directly sequencing the double-stranded products. In a collection of 89 sequenced genes, all types of mutations were found, with transitions (mostly G·C to A·T) constituting the largest class (35%), transversions accounting for 27%, and small deletions/duplications for 25%. Simple base substitutions were distributed throughout the aprt structural gene with few sites having recurring mutations and G·C base-pairs being the predominant substitution target. Small deletions, on the other hand, were not distributed so evenly, being concentrated in a region of aprt rich in short direct and inverted repeat sequences. The base substitutions were predominantly missense, while about 10% produced nonsense codons. Splice junctions, and start and stop codons were also significant targets for mutation. No alterations were detected in three aprt-deficient strains after sequencing all exons and substantial upstream and downstream region.

The structure of the major immediate early gene of human cytomegalovirus strain AD169

The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5′ end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.

RAPID DETERMINATION OF MOLECULAR RELATEDNESS OF CLINICAL CYTOMEGALOVIRUS STRAINS

Restriction enzyme digestion analysis of cytomegalovirus (CMV) isolates is used as a powerful tool to study the epidemiology of CMV infections. Use of this technique is hampered by the necessity of growing sufficient virus in tissue culture which may take 3-6 months. Using 32 EcoRI clones from the AD169 strain of CMV, our studies of the CMV genome show that polymorphism is present in the fragments closest to the long and the short inverted repeat sequences of the joint regions. Complete heterogeneity is present for all different CMV strains when hybridized to joint pieces, F and H. Using these observations, we developed a rapid method to determine molecular relatedness of different clinical isolates of CMV. DNA is extracted directly from primary cultures or clinical specimens containing CMV, cleaved with restriction enzyme EcoRI, separated by electrophoresis, transferred to nitrocellulose filters, hybridized to 32P-labeled fragment F or H, and exposed to x-ray film. Using this procedure, we find that all random isolates of CMV exhibit different fragments hybridizing to clones F and H, while isolates showing identical restriction enzyme digestion patterns exhibit identical DNA bands hybridizing to the joint fragments. This procedure reduces the need to passage CMV in tissue culture, can be performed in less than a week, and should greatly simplify studies designed to define the epidemiology of CMV infections.

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S-16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3'-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.

Coordinately expressed chorion genes of Bombyx mori: is developmental specificity determined by secondary structure recognition?

Short inverted repeat sequences have been observed in the DNA flanking the 5' and 3' termini of a pair of coordinately expressed chorion structural genes of the silkmoth Bombyx mori. When superhelical cloned DNA containing the two chorion genes is digested with S1 nuclease, several sites are specifically cleaved including those around the centers of the putative cruciform structures resulting from the short inverted repeat sequences. The possible implication of conformational changes in the DNA surrounding the chorion structural genes in the process of determination of the developmental specificity and the coordinate transcriptional regulation of these genes is discussed.