Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

Exogenous thymidine 5''-monophosphate as a precursor for DNA synthesis in yeast

Summary Strain MB1015-5C of Saccharomyces cerevisiae can utilize exogenous thymidine 5-monophosphate (5-dTMP) for its DNA synthesis. Studies with either [P³²] or [2-C¹⁴] labelled 5-dTMP reveal first that some of the precursor molecules are taken up intact in DNA synthesis and secondly that 3-digests of highly purified [P³²] DNA yield up to 94% of all [P³²] as 5-dTMP [P³²]. Under the conditions used in these experiments more than 90% of the exogenously supplied 5-dTMP is broken down into orthophosphate and thymidine by an acid phosphatase. Only the orthophosphate is utilized by the yeast cells, mainly for RNA synthesis, and thymidine is not taken up. Suppression of the phosphatase activity is possible by addition of inorganic phosphate to the medium; under these conditions breakdown of 5-dTMP is suppressed but uptake and incorporation of the molecules into the DNA of strain MB1015-5C is still not very effective.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Determination of internal dynamics of deoxyriboses in the DNA hexamer d(CGTACG)2 by 1H NMR

The conformations and internal dynamics of the deoxyriboses of d(CGTACG)₂ have been determined by NMR measurements at 15°C. The conformations of the sugars were determined using coupling constants and time-dependent NOE measurements. The J-splitting patterns of the H1, H2 and H2 resonances show that the sugars exist as mixtures of conformations near C2 endo (south) and C3 endo (north). The population of the south conformation was larger for the purines than for the pyrimidines. The overall tumbling time of the molecule in ²H₂O was determined from measurements of the cross relaxation rate constant for the H6-H5 vectors of the two cytosine residues. Order parameters were determined for the H1-H2, H2-H2 and H2-H3 vectors from measurements of cross relaxation rate constants, making use of multi-spin analysis of the NOE build up rates. These order parameters are weakly dependent of the base sequence, and except for the terminal Cyt 1 residue, the H2-H2 and H2-H3 vectors are near unity, indicating the absence of rapid pseudorotation on the nanosecond time scale. However, the order parameter for the H1-H2 vector is significantly smaller than expected for rapid pseudorotation indicating the presence of other motions of the sugars. This motion must be about an effective axis parallel to the H2-H vector, and to occur with an angular fluctuation of about 30°.The results show that to obtain highly refined structures for nucleic acids by NMR the effects of spin diffusion and motional averaging cannot be ignored.Some of this work was presented as a poster at the 30^th Experimental NMR Conference at Asilomar, California 1989     

Exon shuffling in anther-specific genes from sunflower

We describe here the nucleotide sequence of an anther-specific gene (sf18) from sunflower, encoding a proline- and glycine-rich polypeptide with a hydrophobic amino-terminus (signal peptide). The gene is split by a 211 by intron and is partially related to another anther-specific gene (sf2) from sunflower with which it shares important sequence stretches in the 5 coding and upstream regions. We propose that the two genes have originated via exon shuffling, during which a copy of a DNA segment including the promoter region as well as a signal peptide coding sequence has been transferred into the upstream region of two different potential coding sequences, generating two novel genes which display the same specificity of expression and which both encode an extracellular protein. While the 5 region of the intron is highly conserved as part of the transferred region and may play a role in the selection of the 5 splice site, a common octanucleotide at the 3 end of the intron of the two genes might be involved in 3 splice site selection.     

The L1 family of long interspersed repetitive DNA in rabbits: Sequence,copy number,conserved open reading frames,and similarity to keratin

Summary The L1 family of long interspersed repetitive DNA in the rabbit genome (L1Oc) has been studied by determining the sequence of the five L1 repeats in the rabbit -like globin gene cluster and by hybridization analysis of other L1 repeats in the genome. L1Oc repeats have a common 3 end that terminates in a poly A addition signal and an A-rich tract, but individual repeats have different 5 ends, indicating a polar truncation from the 5 end during their synthesis or propagation. As a result of the polar truncations, the 5 end of L1Oc is present in about 11,000 copies per haploid genome, whereas the 3 end is present in at least 66,000 copies per haploid genome. One type of L1Oc repeat has internal direct repeats of 78 bp in the 3 untranslated region, whereas other L1Oc repeats have only one copy of this sequence. The longest repeat sequenced, L1Oc5, is 6.5 kb long, and genomic blot-hybridization data using probes from the 5 end of L1Oc5 indicate that a full length L1Oc repeat is about 7.5 kb long, extending about 1 kb 5 to the sequenced region. The L1Oc5 sequence has long open reading frames (ORFs) that correspond to ORF-1 and ORF-2 described in the mouse L1 sequence. In contrast to the overlapping reading frames seen for mouse L1, ORF-1 and ORF-2 are in the same reading frame in rabbit and human L1s, resulting in a discistronic structure. The region between the likely stop codon for ORF-1 and the proposed start codon for ORF-2 is not conserved in interspecies comparisons, which is further evidence that this short region does not encode part of a protein. ORF-1 appears to be a hybrid of sequences, of which the 3 half is unique to and conserved in mammalian L1 repeats. The 5 half of ORF-1 is not conserved between mammalian L1 repeats, but this segment of L1Oc is related significantly to type II cytoskeletal keratin.     

A site-specific endonuclease from Pseudomonas aeruginosa

Pael, a new restriction endonuclease from Pseudomonas aeruginosa clinical strain was isolated and characterized. It recognizes and cleaves the sequence 5-GCATGC-3 generating DNA fragments with 3-tetranucleotide sticky ends. DNAs of pBR322, SV40 and bacteriophage have one, two and six Pael recognition sites, respectively.Seventytwo strains of Pseudomonas, Clostridium, Escherichia coli, Shigella, Proteus and Saccharomyces were screened for the presence of site-specific endonucleases. Here we describe the Pael restriction enzyme found in Pseudomonas aeruginosa; other data will be published elsewhere.Earlier Hinkle and Miller isolated from P. aeruginosa a PaeR7 restriction endonuclease recognizing and cleaving a sequence 5-CTCGAG-3 (1). Sequence analysis of DNAs cleaved by PaeI shows that the enzyme is the isoschizomer of SphI (2).     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects

Summary The sequences of a collection of 261 spontaneous lacI^- mutants recovered in a PolA^- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5-GTGG-3 sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5-GTGG-3 sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5-GTGG-3 sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.     

Polarity of DNA entry in transformation of Streptococcus pneumoniae

Summary DNA transport in Streptococcus pneumoniae was studied using donor molecules labelled either at the 3 or at the 5 end, on one strand only. In contrast to 5 end label, 3 end label was not taken up by the cells indicating that entry is a polarized process. Our results together with those of previous studies are consistent with a model for entry in which double-stranded donor DNA is nicked on binding at the cell surface. Entry of a single strand then proceeds linearly from a newly formed 3 end to the extremity of the donor fragment.