Journal of molecular and applied genetics最新文献

A strain of Saccharomyces cerivisiae carrying a mutation in the URA1 gene was transformed with fragments of Dictyostelium DNA inserted into a plasmid capable of replication in E. coli or yeast. Rare prototrophic colonies were recovered that all contained the parent plasmid and an insert of Dictyostelium DNA. Resistance to the antibiotic G418, a function coded by the same plasmid, was also expressed in the prototrophs. Plasmids recovered from the prototrophic yeast could be used to transform E. coli. The E. coli transformants harbored plasmids capable of transforming yeast URA1 mutants to prototrophy. No complementation of the E. coli pyrD mutation, which corresponds to URA1, occurred. Southern blot analysis revealed that the insert of Dictyostelium DNA contained a unique sequence of 1700 base pairs and a repetitive one of 1000 base pairs. Subcloning experiments showed that only the unique sequence was required for complementation, which is independent of the orientation of the Dictyostelium sequence in the plasmid. The repetitive fragment was not linked to the unique sequence in the genome and was probably an artifact of the ligation procedure. The unique sequence hybridized to a Dictyostelium polyA+ RNA species of 1200-1300 bases.

BamHI decanucleotide linkers (5' CCGGATCCGG 3') were ligated to full-length linear polyoma DNA partially cleaved with HincII, and the recombinant DNA was transfected into mouse 3T6 cells. A viable virus (PYNB5) was isolated which contains four linkers at the 26-min HincII site. PYNB5 is encapsidated with a larger major viral capsid protein (VP1) as predicted from the DNA sequence. PYNB5 appears to have the same growth and physical properties as the wild-type virus with the exception of greater inactivation upon dilution of virus stocks with water. When PYNB5 DNA is cleaved with BamHI, the larger of the two resulting fragments (PY66) contains an intact early region of the virus, including the origin of DNA replication. PY66 complements a polyoma early region mutant for growth and may be useful as a cloning vector for foreign DNA.

The DNA sequence of the nopaline synthase gene (nos) from Agrobacterium tumefaciens Ti plasmid pTiT37 and adjacent regions up to the right border of the T-DNA was determined. The 5' and 3' termini of the polyadenylated nos mRNA, isolated from a T37 tobacco teratoma tumor line, were localized by S1 mapping. The final mRNA is unspliced, encoded by a region of about 1450 bp, and specifies an open reading frame of 413 amino acids. Potential transcriptional signals in the 5' flanking DNA, such as CATAAA ("TATA box") and GGTCACTAT ("CAT box"), bear close resemblance to other eukaryotic promoters. Two putative polyadenylation signals, AATAAA and AATAAT, are found about 135 and 50 bp from the 3' end, respectively. This study may provide information for the development of expression vectors for genes in plant cells; moreover, the structural gene can be used as an easy screenable marker.

Hybridization studies carried out with poly(A)+ RNA and its corresponding cDNA showed the presence of a new highly abundant RNA class after heat shock (hs) at 40 degrees C in soybean hypocotyl compared to tissue incubated under normal conditions at 28 degrees C. cDNA clones complementary to RNAs of this class were isolated; eleven clones were characterized and used in the analysis of these abundant RNAs. The most abundant hs-sequences were found to be 800-900 nucleotides in length and present in about 19,000 copies per cell. Extensive sequence homology among hs-induced RNAs was indicated by cross-hybridization of cDNA clones and by common protein patterns generated in hybrid release translations. The existence of at least two different nucleotide sequences common to several different hs poly(A)+ mRNAs was documented by different, nonoverlapping protein patterns obtained by in vitro synthesis with hybrid selected RNAs. Four clones contained a sequence common to mRNAs for at least 13 proteins of 15,000-18,000 daltons; another sequence common to mRNA for three to four proteins of 21,000-23,000 daltons was selected by one clone. Two other clones selected a major hs-protein of about 18,000 daltons. The mRNAs of these low molecular weight hs-proteins accumulated rapidly after induction at either 40 degrees C or 42.5 degrees C and decreased rapidly during subsequent incubation at 28 degrees C.

Molecular cloning has been used to isolate the ends of that portion of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which has been designated T-DNA and which has been transferred to the genome of tobacco crown gall tumor cells. Analysis of the DNA sequences of the plant border clones compared with the corresponding sequences of the Ti plasmid suggests that the mechanism of transferred DNA integration and subsequent stabilization is precise at the right border and imprecise on the left. The T-DNA junction occurs within a variation of a single base pair (bp) on the right but varies over at least 70 bp on the left. In addition, there are several sequences which are repeated near the ends of the T-DNA region in the Ti plasmid. Seemingly, there is no specificity with regard to the site of integration in the plant genome.

We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.

Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain. T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes. This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned. Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized.

Seven mutations that affect various activities of the multifunctional DNA binding protein (DBP) encoded by human adenovirus have been physically mapped to different locations within DBP gene by marker rescue experiments. Two of these mutants (Ad5ts107 and Ad5ts125) contain a lesion which, under nonpermissive temperatures, decreases the capacity of the protein to bind single-strand DNA, blocks DNA replication, and prevents normal turn-off of viral early genes. In addition, the efficiency of transformation by the ts viruses compared to wild-type virus is increased at the nonpermissive temperature. Both ts mutations are located in the 3' half of the DBP gene (C-terminal half of DBP). In contrast, the alterations in the five host range mutants (Ad2hr400-Ad2hr403, Ad5hr404) which overcome the block to viral late mRNA synthesis in monkey cells, but have no marked effect on DNA replication or early gene expression map in the 5' half of the DBP gene. These results suggest that the 72 kd DBP of adenovirus contains at least two functionally separable domains.

We constructed hybrid plasmids to allow controlled expression of the lpp gene coding for the outer membrane lipoprotein of Escherichia coli, which is otherwise expressed constitutively. This was achieved by the insertion of a DNA fragment carrying the lacUV5 promoter-operator region as a transcriptional control switch into the 5'-untranslated region of the lpp gene. When fully induced, the production of the lipoprotein, controlled under the tandem promoters of lppp-lacpo-lpp, increased approximately 3-fold compared to that under lacpo-lpp control. However, it was still only one-third of the lipoprotein production under the constitutive lpp expression. One such plasmid, pKEN125, carrying lppp-lacpo-lpp in pBR322 produced only a trace amount of the lipoprotein without induction in an E. coli lpp- cell. Upon the addition of isopropyl-beta-d-thiogalactoside, however, the amount of the lipoprotein reached almost 40% of the total membrane proteins. Cells carrying pKEN125 grew normally in the presence of the inducer, whereas cells carrying plasmid pKEN126 with tandem duplication of lppp-lacpo-lpp sequences in pBR322 lysed upon induction at high temperature. In cells with pKEN126 induced at high temperature, at least three new bands which were cross-reactive with antilipoprotein serum in addition to the mature lipoprotein were detected by pulse-labeling cells with [35S]methionine.

During third-instar larval development of Drosophila melanogaster, the fat body tissue synthesizes six major methionine-containing polypeptides, three of which are the alpha, beta, and gamma subunits of the hexameric larval serum protein LSP-1, a fourth is the single subunit of the hexameric larval serum protein LSP-2, and the other two are polypeptides P6 and P1. Genomic DNA clones of the six structural genes for the polypeptides were isolated and characterized. Each gene maps by in situ hybridization at a single chromosomal site and appears to be present as a single copy in the genome. The LSP-1 and LSP-2 genes show striking regulatory similarities: The LSP-1 beta and gamma transcripts are first detected in fat bodies within an hour after the second molt, and the LSP-1 alpha and LSP-2 transcripts a few hours later; the four transcripts are subsequently maintained at high levels during most of the third instar and rapidly decrease shortly before pupariation. Ecdysterone increases the levels of at least three of the four LSP transcripts in the fat bodies when ecdysterone-deficient larvae from the temperature-sensitive mutant ecd1 are supplemented with the hormone. The regulatory characteristics of the P6 and P1 genes differ in several ways from those of the LSP genes. Expression of the P6 and P1 genes begins later than the LSP genes, and the levels of the transcripts remain high at the end of the third instar after the LSP transcripts have markedly decreased. Ecdysterone increases the level of the P1 transcript, but not of the P6 transcript, in ecdysterone-deficient ecd1 larvae.