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PubMed1978

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.

Base Sequence↗
PubMed1980

Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions.

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

Base Sequence↗
PubMed1981

The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis.

We propose that the initial event in the secretion of proteins across membranes and their insertion into membranes is the spontaneous penetration of the hydrophobic portion of the bilayer by a helical hairpin. Energetic considerations of polypeptide structures in a nonpolar, lipid environment compared with an aqueous environment suggest that only alpha and 3(10) helices will be observed in the hydrophobic interior of membranes. Insertion of a polypeptide is accomplished by a hairpin structure composed of two helices, which will partition into membranes if the free energy arising from burying hydrophobic helical surfaces exceeds the free energy "cost" of burying potentially charged and hydrogen-bonding groups. We suggest, for example, that the hydrophobic leader peptide found in secreted proteins and in many membrane proteins forms one of these helices and is oriented in the membrane with its N terminus inside. In secreted proteins, the leader functions by pulling polar portions of a protein into the membrane as the second helix of the hairpin. The occurrence of all categories of membrane proteins can be rationalized by the hydrophobic or hydrophilic character of the two helices of the inserted hairpin and, for some integral membrane proteins, by events in which a single terminal helix is inserted. We propose that, because of the distribution of polar and nonpolar sequences in the polypeptide sequence, secretion and the insertion of membrane proteins are spontaneous processes that do not require the participation of additional specific membrane receptors or transport proteins.

Biological Transport↗
PubMed1981

Structure and evolution of goat gamma-, beta C- and beta A-globin genes: three developmentally regulated genes contain inserted elements.

We have determined the complete nucleotide sequence of the goat fetal (gamma), preadult (beta C) and adult (beta A) globin genes. In contrast to other globin genes expressed in different stages of development, these three genes are highly homologous (approximately 90%) in both their coding and noncoding regions. The only major difference between them results from elements inserted into their large introns: gamma contains a 247 bp insertion, beta A contains a 318 bp insertion, and beta C contains both a 252 bp insertion and a 60 bp deletion. Based on comparisons of these three genes to each other and to two goat globin pseudogenes, psi beta X and psi beta Z, we conclude that gamma, beta A and beta C have diverged relatively recently, and that the two gene pairs psi beta X - beta C and psi beta Z-beta A arose via a block duplication of an ancestral pseudogene-functional gene pair. Moreover, as opposed to the human fetal genes (G gamma and A gamma), the goat gamma gene appears to be a true beta-like gene that has fetal-specific function. The insertions in gamma, beta A and beta C contain sequence features characteristic of transposon-like elements, and appear to be represented in multiple copies throughout the genome.

Animals↗
PubMed1987

Fertility effects of chromosome rearrangement (insertion 16) in Charolais cattle from Brazil.

Insertion 16, a structural chromosome abnormality originated by three breaks in one autosome, was detected with familial recurrence in a Charolais cattle population. Due to the peculiar nature of Insertion 16, its effect on the fertility of the carriers was verified by various parameters. A total of 115 animals was karyotyped to learn the segregational behavior of the chromosome abnormality, and 1,354 females were studied to evaluate the reproductive efficiency of the propositi, a pair of chimeric bulls for insertion 16. Their fertility was observed and compared with that of three normal bulls. Analysis of the results revealed that insertion 15 segregates in the offspring of only one heterozygous proposita, without sex preference, and that the percentage of conceptions in the cows inseminated by the propositi was similar to the controls'; however, for each conception with the semen of the propositus in which the insertion segregated, 0.16 more inseminations were necessary than for the controls. Similarly, the deviation in the percentage of nonreturns after the first insemination (15.9%) and the frequency of abnormal oestrous cycles (10.3%) indicate reduced fertility in the propositus, which is related to early embryo mortality.

Journal Article↗
PubMed1985

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid.

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.

Bacterial Proteins↗
PubMed1988

A novel insertion sequence in the cryptic plasmid of Neisseria gonorrhoeae may alter the B protein at the translational level.

A variant of the cryptic plasmid of Neisseria gonorrhoeae, 4.4 kb in size, was isolated and characterized at the molecular level. This variant harbored a 156-bp insertion which was located between coordinates 3134 and 3135 within the putative cppB gene using the 4.2-kb cryptic plasmid, pJD1, as a reference. The insertion contained a novel EcoRI site and several elements of symmetry (both direct and inverted repeats). Stop codons present in the insertion interrupted the coding capacity of the cppB gene. Although the insertion was within one of two previously characterized 44-bp repeats purportedly involved in site-specific recombination, it was distinct from a 54-bp segment deleted in some cryptic plasmids. The presence of the insertion suggests a mechanism of modulating the expression of the cppB gene at the translational level through DNA rearrangement.

Amino Acid Sequence↗
PubMed1985

The primary structure of the rRNA insertions of Plasmodium lophurae.

The DNA sequences of the novel insertion in the 17s rRNA gene and the large insertion in the 25s rRNA gene in the cloned rDNA unit of the avian malaria parasite Plasmodium lophurae are presented, together with a partial sequence of the flanking regions, which code for the mature rRNA. The homology of the mature rRNA coding regions with the rRNA sequences of other eukaryotic organisms is extensive, indicating that the plasmodium rRNA is structurally similar to other eukaryotes. Sequence data also reveal that the region 3' to the insertion in the 17s rRNA contains a second small inserted DNA sequence, in contrast to other known small rRNA sequences. The region containing the 25s insertion shares sequence homology and some secondary structure characteristics with the terminal direct repeat of the Drosophila melanogaster transposable element copia. This is the first such sequence described in plasmodia. The direction of transcription of the cloned rDNA unit of P. lophurae has also been determined. As in other organisms, the direction of transcription is found to be 5' 17s-25s 3'.

Animals↗
PubMed1994

Analysing nuclear shape as a function of relative spatial position in the femoral insertion of the medial collateral ligament.

Quantification of biological structure by morphometry facilitates the correlation of structure to biological function. To test a hypothesis concerning the correlation of a gradient in cell and nuclear shape and a corresponding gradient in mechanical stress within a ligament insertion into bone, a computerized approach was developed for quantifying mean nuclear shape as a function of position within this insertion. Three femoral insertions of the medial collateral ligament of the rabbit were prepared for histology. Commercially available software was used to measure nuclear perimeter and area by video-based planimetry on microscopic images, then custom software was used to define an overlying mesh of polygons on each insertion, to normalize the geometry of the insertion, and to calculate the mean nuclear roundness (a function of area and perimeter) for each polygon in the mesh. This approach allows the comparison of mean nuclear roundness of different polygons and among different animals that would be extremely difficult to do manually. In addition, these morphometry results can be used to correlate with functional data.

Animals↗
PubMed1995

A unique insertion in the S gene of surface antigen--negative hepatitis B virus Chinese carriers.

The presence of unique hepatitis B virus (HBV) variants has been investigated in two Chinese patients with chronic liver disease, whose sera were positive for HBV-DNA by dot blot hybridization or polymerase chain reaction (PCR) but hepatitis B surface antigen (HBsAg)-negative by conventional polyclonal antibody based immunoassays. PCR amplification of HBV-DNA followed by direct sequencing showed an insertion of six nucleotides, which introduced two additional amino acids between codons 122 and 123 in one patient (Isolate 1), whereas a nine nucleotide insertion in the other patient (Isolate 2) gave rise to three amino-acids between codons 123 and 124 immediately upstream from the 'a' determinant in the S gene. These insertions have not been described previously in any published sequences of the known subtypes and were absent from sequences of 30 HBsAg-positive Chinese patients from the same region. In the cases under study, the insertion is associated with four consecutive adenine molecules from nucleotides 516 to 519. It seems likely that this area is a hot spot for insertions in HBV. We found none of the previously described amino-acid deletions or substitutions in the pre-S1, pre-S2 and S genes, which are involved in unusual antigenic profiles. This finding suggests that genetic mutations in the S gene outside the 'a' determinant may be responsible for failure to detect HBsAg in some Chinese patients with chronic hepatitis caused by HBV infection.

Adult↗
PubMed1987

Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM).

Two cDNA inserts complementary to mRNA encoding aromatase cytochrome P-450 (P-450AROM) have been isolated and characterized by restriction mapping and sequencing. The overlapping sequence encoded by these inserts is identical, and a putative heme-binding region has been identified. In addition, the open reading frame contains the sequences of all four cysteine-containing tryptic peptides isolated by Chen et al. (1986) from purified cytochrome P-450AROM. The inserts differ in the use of alternative poly A-addition signals, which is consistent with the presence of two major species of mRNA in human placenta, of 3.0 and 2.4 kb, which hybridize to these inserts. The identity of sequence between the two inserts and the likely presence of alternative poly A-addition signals, is suggestive that only one form of cytochrome P-450AROM is encoded by these mRNA species.

Amino Acid Sequence↗
PubMed1979

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.

Bacteriophage lambda↗
PubMed1985

Nonrandom insertion of Tn5 into cloned human adenovirus DNA.

The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition. To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis. We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition. However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion. We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1. Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences. Our data suggest that transcriptional activity may influence Tn5 transposition.

Adenoviruses, Human↗
PubMed1985

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering.

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.

Cloning, Molecular↗
PubMed1986

Construction of recombinant vaccinia virus strains using single-stranded DNA insertion vectors.

The ability of single-stranded (ss) DNA, isolated from recombinant M13 bacteriophage, to direct the insertion of foreign genetic elements into the vaccinia virus (VV) genome was examined. An identical chimeric transcriptional unit [VV promoter/chloramphenicol acetyl transferase (CAT) gene embedded in DNA sequences encoding vaccinia virus thymidine kinase (TK)] was inserted into either the previously characterized plasmid insertion vector, pGS20, or into M13mp18. It was found that the ss vector (M13mp18:TK/CAT) was four times more efficient than the plasmid vector (pGS20:CAT) in catalyzing homologous recombination of the cat gene by marker transfer into the VV genome. Furthermore, Southern blot analyses and CAT enzymatic activity assays confirmed that the structure of the M13-derived recombinant genomes were as expected and that the chimeric genes were fully active. Although the precise mechanism responsible for the ss DNA-catalyzed insertion event is not known, these results are discussed with respect to the advantages of using M13-based vectors with which to manipulate and insert genetic information into infectious VV recombinants.

Cloning, Molecular↗
PubMed1987

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria.

We have constructed a series of derivatives of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these omega derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an omega interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of omega insertional mutations can be carried out in these bacterial species.

DNA Restriction Enzymes↗
PubMed1990

Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum.

Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions. However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends. During the study of this phenomenon, we obtained KmR Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA. Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region. The most conspicuous result from Southern analyses on four such KmR plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence. Four truncated T-DNA insertions have been cloned. Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA. Another is missing the left T-DNA part and the last T-DNA is lacking both ends. Sequence analysis of the T-DNA::plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence. S1-type mapping of the most strongly expressed plant genome::nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic Pnos-nptII plants.

Base Sequence↗
PubMed1992

The 28S ribosomal RNA-encoding gene of Hymenoptera: inserted sequences in the retrotransposon-rich regions.

The genomes of two parasitoid wasps, Diadromus pulchellus and Eupelmus vuilleti, and the honey bee, Apis mellifera, contain few interspersed repeated sequences corresponding to transposons (Tn). This suggests that the genomic organisation of Hymenoptera could be due to the elimination of deleterious Tn in haploid males. We have used restriction-fragment length polymorphism analysis to show that nondeleterious Tn are present in the DNA (rDNA) encoding ribosomal RNA of twelve species of Hymenoptera. Sequence analysis of the 28S rDNA type-I and type-II insertion-rich regions of 80 species showed that this region is very highly conserved (95.8%). A consensus sequence and restriction map of the rDNA region were established. These sequence data were used to develop a strategy for detecting inserted elements in the rDNA fragments containing type-I or type-II insertion sites, and this strategy was used to screen twelve hymenopteran species and four non-Hymenoptera control species. The rDNA fragments from the Hymenoptera and control species contained inserted sequences in the area where type-I and type-II elements are inserted in the 28S rDNA retrotransposon-rich region of Diptera and Lepidoptera. The hymenopteran genomes therefore appear to contain repeated elements, the mobility and nature of which remain to be determined.

Animals↗