The biosynthesis and nutritional uses of carotenoids.

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character No highly conserved 'homology blocks' are found in either class of topogenic sequence Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14679.x

Domain structure of mitochondrial and chloroplast targeting peptides.

The ability to attach to epithelial cells, efface the microvillus surface, and disrupt the underlying cytoskeleton is characteristic of enteropathogenic Escherichia coli (EPEC). Recently, eae, a gene necessary for this phenomenon, was described (A. E. Jerse, J. Yu, B. D. Tall, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 87:7839-7843, 1990). We report the use of a novel suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis to construct an eae deletion mutant of EPEC. This system enables positive selection for the loss of vector sequences. The resulting mutant, CVD206, is indistinguishable from the wild-type strain except for the loss of a 94-kDa outer membrane protein and attaching and effacing ability. Both the 94-kDa outer membrane protein and attaching and effacing ability are restored upon reintroduction of the eae gene on a plasmid. These results confirm the role of the eae gene in the attaching and effacing activity of EPEC and establish the utility of a new system for the construction of deletion mutations.

https://iai.asm.org/content/iai/59/12/4310.full.pdf

Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system.

pACYC184 ii a commonly used multicopy cloning vector which was constructed by ligating restriction fragments from pSClOl, Tn9, and pl5A each of which have been previously sequence*! (1,23,4,5). Despite its wide use, the complete nucleotide sequence of pACYC 184 has never been repented. The sequence was completed by using oligonudeotide primers designed to span the junctions between each of the previously sequenced regions. pACYC184is 4244 bp in length with nucleotide 1 corresponding to the EcoRI site in the original map (1). The chtorampbenicol resistance (Cm) segment from Tn9 extends from the Haell site at base 3505 to the Haell site at base 585 with bases 219 (ATG) to 3804 encoding the Cm gene. Part of an IS1 (5) from Tn9 extends from bases 443 to 583. Bases 1494 to 3275 are derived from pSClOl with the tetracycline (Tet) resistance gene encoded by bases 1580 (ATG) to 2770. The pl5A origin of replication extends from bases 581 to 1492. Three fragments; an Alul (3276) to Haell (3368), a Haen (3368) to Haell (3422), and a Haell (3422) to Haell (3505) are located between Cm and Tet gene and are all derived from different regions of the Tet gene. During the construction of pACYCl84 a precursor plasmid, pACYC175, was digested with Haem, Alul, and Hindi, to remove extraneous DNA and to reduce the size of the plasmid. It appears that the precursor was also digested with Haell which generated the Haell Cm resistance segment and the Haell fragments found between the Tet and Cm genes. The underlined sequence was determined while the rest of the sequence was taken from the published sequences of pSClOl (2), Tn9 (3,4), and pl5A (5).

The nucleotide sequence of pACYC184

pUC4K Is a vector that carries the kanamycin resistance determinant from Tn903 (1) and has been used not only in the construction of new vectors but also as a restriction mobilization element to create codon Insertions of varying length and location In cloned genes (1). pUC4K was originally constructed by cloning a 1.4 kb Haell fragment from Tn903 Into the Pstl site of pUC7 by G-C tailing (1). The kanamycin resistance gene contained within the Haell fragment should be flanked by 226 bp Inverted repeats (2), but in the process of constructing pUC kanamycin derivatives we have discovered that one of these Inverted repeats is missing. By sequencing through each of the Q-C tails Into the kanamycin resistance determinant we found that the inverted repeat at the 5* end of the gene had been entirely deleted except for 6 bp. Initially we thought this deletion was unique to our Isolate of pUC4K, but pUC4K from other sources (Pharmacia, colleagues) contained the same deletion. Thus, the Tn903 fragment contained in pUC4K extends from bases 1052 to 2264 instead of bases 831 to 2264 as originally suggested (2). The deletion appears to have occured during the initial construction of pUC4K, perhaps by tailing at a nick at base 1052.

/pdf/a-correction-in-the-nucleotide-sequence-of-the-tn903-v3f5fx6d6x.pdf

A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K

pACYC177 is a commonly used multicopy cloning vector (1), however, its complete nudeotide sequence has never been reported. The plasmid was constructed by ligating restriction fragments from three different sources: Tn903, Tn3, and pl5A each of which has been sequenced (2,3,4). The sequence was completed by using oligonucleotide primers designed to span the junctions between each of the previously sequenced regions. pACYC177 is 3940 bp in length with nucleotide 1 corresponding to the HincII site in the original map (1). The Tn3 region extends from bases 3026 to 756 with the beta-lactamase gene encoded by bases 3698 (ATG) to 615. Part of the Tn3 repressor extends from bases 3025 to 3515, but the promoter region and part of the coding region have been deleted. In addition, one inverted repeat from Tn3 extends from bases 728 to 765. The Tn903 region, bases 1595 to 3025, contains the kanamycin resistance determinant which extends from bases 1922 (ATG) to 2734 flanked by 226 bp inverted repeats. The pl5A origin of replication extends from bases 766 to 1595. The underlined sequence was determined while the rest of the sequence was taken from the published sequences of Tn9O3 (2), Tn3 (3), and pl5A (4).

The nucleotide sequence of pACYC177

An expressed napin storage protein gene from Brassica rapa, BcNA1, has been cloned and sequenced. The gene is a member of a family of four to seven napin genes in B. rapa and is highly expressed in developing seeds. An expression cassette containing the DNA flanking the napin coding region of BcNA1 has been engineered and demonstrated to function appropriately, as compared with the gene's endogenous expression, in transgenic rapeseed using the β-glucuronidase reporter gene. The B. rapa BcNA1 gene and a B. napus napin gene, gNa, share extremely high nucleotide homology not only throughout their coding regions, but over a DNA locus comprising 4.3 kb. We suggest the gNa gene was contributed by the original B. rapa progenitor of the amphidiploid B. napus.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/6B17B63529ECD38D235CF85402F846A0/S0960258500000921a.pdf/div-class-title-isolation-and-characterization-of-an-expressed-napin-gene-from-span-class-italic-brassica-rapa-span-div.pdf

Ronald E. Rose

Papers

The nucleotide sequence of pACYC184

A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K

The nucleotide sequence of pACYC177

Isolation and characterization of an expressed napin gene from Brassica rapa

The nucleotide sequence of a cDNA clone encoding acyl carrier protein (ACP) from Brassica campestris seeds.