Comparison of the sequences at specific sites on DNA cleaved by the antitumor antibiotics talisomycin and bleomycin.

We have investigated the site-specific cleavage of DNA by the antitumor antibiotics talisomycin and bleomycin by using 5'- or 3'-terminal 32P-labeled restriction fragments of pBR 322 DNA. Both drugs cleaved DNA preferentially at G-C and G-T sequences. However, the relative amounts of cleavage at particular cleavage sites differed between talisomycin and bleomycin at concentrations of the drugs which produced similar extents of total cleavage. In addition, talisomycin produced specific cleavages at G-A sequences which were relatively resistant to cleavage by bleomycin. Within a preferred sequence group (i.e., G-C sequences), some sites were cleaved to a greater extent relative to others by both talisomycin and bleomycin, suggesting that a greater degree of specificity than that provided by only two nucleotides is involved in the site-specific recognition and cleavage of DNA by these drugs.

Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3',5'-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5' genomic DNA.

The complete nucleotide sequence for rat ovarian aromatase cytochrome P450 (P450arom) has been derived from four cDNA clones isolated from three rat granulosa/luteal cell lambda gt11 cDNA expression libraries. The composite P450arom cDNA extends 1597 basepairs, encodes a protein of 508 amino acids (calculated mol wt = 58,263), and hybridizes to three mRNA transcripts (3.3, 2.6, and 1.9 kilobases in size) in rat ovarian tissues. A 5' genomic fragment was isolated from a rat genomic library and shown to contain exon I and 538 basepairs of 5' flanking sequences, including putative promoter elements. Further, we document that P450arom mRNA and estradiol (E) biosynthesis are regulated by cAMP-dependent mechanisms in granulosa cells of preovulatory (PO) follicles, but are maintained by cAMP-independent mechanisms after LH/hCG-induced luteinization. The transition of the PO granulosa cell to the luteal cell (PO + hCG) phenotype requires 5 h of exposure to hCG in vivo. Once the luteal cell phenotype is programed, P450arom mRNA and E biosynthesis are maintained in the luteinized cells for up to 10 days in a constitutive manner in the absence of hormones or agents that increase intracellular cAMP. Furthermore, when PO + hCG (7 h) follicles were isolated and incubated for 1-3 h with reversible inhibitors of transcription (actinomycin-D) or translation (cycloheximide) before harvesting the granulosa cells, neither morphological nor functional luteinization of granulosa cells in culture was impaired. Thus, rapid cellular and molecular events occur in granulosa cells within 5-7 h after an ovulatory LH/hCG surge that alter the hormonal regulation of the aromatase gene.

Sequence and expression of a functional chicken progesterone receptor.

We have cloned and sequenced 4.5 kilobases (Kb) of cDNA encoding the chicken progesterone receptor. The complete cDNA contains an open reading frame of 2361 nucleotides in length and encodes a polypeptide of 787 amino acids with a mol wt of 85.9 K. At least four mRNA species have been detected in chick oviduct cells. Direct sequencing of variant cDNAs has suggested that two of the mRNAs (4.5 Kb and 3.6 Kb) differ only in the length of their 3'-untranslated regions. A third mRNA (1.8 Kb) produces a truncated polypeptide which encodes the immunoreactive NH2 terminal sequence of the receptor but lacks the hormone binding regional and half of the DNA-binding domain. The polypeptide expressed from the receptor cDNA in progesterone receptor negative Cos M-6 cells is indistinguishable from oviduct progesterone receptor in terms of hormone binding and antibody reactivity. Furthermore, the cloned receptor is capable of activating transcription of a target gene. This activation is progesterone dependent (with half-maximal stimulation at approximately 3.3 x 10(-10) M) and specific for the target gene.

Structure and evolution of human alpha-fetoprotein deduced from partial sequence of cloned cDNA.

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human alpha-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% (56/128) amino acids and 54% (207/384) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.

EcoRI-generated reiterated components of the rat genome. I. Sequence of two (92 and 93 bp) related DNA fragments.

The sequence of the 92 and 93 bp long, highly repetitive DNA fragments, isolated from EcoRI digested rat liver DNA, were determined. These fragments, designated 92 and 93, are found in equal abundance, 6.5 x 10(5) copies per haploid genome. J92 and J93 can be distinguished by their differential sensitivity to cleavage by HaeIII and HindIII, respectively, which cut the fragments at 75 and 57 bp from their mutually homologous 5'-ends. J92 and J93 are 38% and 35.4% G + C, respectively, and contain a disproportionate number of triplets complementary to stop codons in all reading frames. Three methylated sites were found in J92 while none could be detected in J93. The sequences around the m5C sites were 5'-Py-Py-m5C-G-Pu-Pu, except for one case where the second Py was replaced by an A. This site appeared to be hemimethylated. When J92 and J93 are placed in register from their mutually homologous 5'-ends, homology is 73% for the first 30 bp region and 63.5% for the total molecule. Thermal melting studies indicate sequence heterogeneity within J92 and J93 from substantial internal base mismatches. The sequences derived are therefore composite averages for the whole molecules. The Cot1/2 for the sequence was measured spectrophotometrically to be 2 x 10(-2) M/s on a DNA phosphorus basis and 2.15 x 10(-4) M/s on a mole fragment basis.

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apo VLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences.

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellenin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.

Amino acid sequence analysis, gene construction, cloning, and expression of gelonin, a toxin derived from Gelonium multiflorum.

The plant toxin gelonin is an extremely potent inhibitor of protein synthesis, similar in action to ricin. The mature protein primary sequence was obtained using conventional sequencing techniques. Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues. This toxin shares approximately 33% sequence homology with trichosanthin and ricin A chain. A 774 bp synthetic gene encoding gelonin was synthesized and expressed in E. coli. Recombinant gelonin (approximately 28 kD) expression was monitored and demonstrated by western analysis. Purification and functional activity studies demonstrated that this protein behaves identically to that of the natural product. Recombinant gelonin (RG) thus joins a growing list of recombinant toxins currently available for use in the construction of recombinant immunotoxins composed of gelonin fused to binding domains of antibodies, growth factors, or other cytokines.

Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides.

In this article we introduce a strategy of preannealing labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous delta F508 and heterozygous delta F508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, delta I507, delta F508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant alleles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with 32P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base stacking interactions between the "capture probe" and the auxiliary oligonucleotide. The presence of the delta F508 mutation was detected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mer probes was improved by increasing the temperature of hybridization from 15 to 25 degrees C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis: 1. A convenient means of introducing label into the target DNA molecule. 2. Disruption of interfering short-range secondary structure in the region of analysis. 3. Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target). 4. Tandem hybridization with the capture probe (providing contiguous stacking) as a means for achieving efficient mismatch discrimination at the terminal position of the capture probe (adjacent to the auxiliary oligonucleotide). By use of multiple auxiliary oligonucleotides, all of the above benefits can be derived simultaneously.

Mutation detection by stacking hybridization on genosensor arrays.

A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled "stacking oligonucleotide," and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the "capture probe" and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3'-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivatized glass. "Operating parameters" examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1. Optimal mismatch discrimination with 9-mer probes occurs at 45 degrees C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45 degrees C. 2. At 25 degrees C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5'-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied. These results provide a motivation for continued development of the stacking hybridization technique for nucleic acid sequence analysis. This approach offers several advantages over the traditional allele-specific oligonucleotide hybridization technique, and is distinct from the contiguous stacking hybridization sitrategy that the Mirzabekov laboratory has introduced (Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov et al. (1996) Nucleic Acids Res. 24, 2998-3004).

Hybridization of DNA targets to glass-tethered oligonucleotide probes.

Hybridization of nucleic acids to surface-tethered oligonucleotide probes has numerous potential applications in genome mapping and DNA sequence analysis. In this article, we describe a simple standard protocol for routine preparation of terminal amine-derivatized 9-mer oligonucleotide arrays on ordinary microscope slides and hybridization conditions with DNA target strands of up to several hundred bases in length with good discrimination against mismatches. Additional linker arms separating the glass surface from the probe sequence are not necessary. The technique described here offers a powerful tool for the detection of specific genetic mutations.

Bovine interstitial retinol-binding protein (IRBP)--isolation and sequence analysis of cDNA clones, characterization and in vitro translation of mRNA.

Three clones for b-IRBP were isolated by anti b-IRBP screening of two bovine retina libraries in the expression vector lambda gt11. The cDNA inserts were then used as hybridization probes to screen and isolate three more clones in a bovine retina library in the non-expression vector lambda gt10. The six overlapping clones generated a b-IRBP cDNA sequence of 3400 nucleotides. An open reading frame encoded the complete amino acid sequences of 8 of the 35 b-IRBP tryptic peptides purified in the present study. One tentative glycosylation site was identified. The coding region was followed by TAG translation terminating codon and an untranslated stretch of about 1700 nucleotides that ended in a sequence containing a presumptive AATAAA polyadenylation signal that was 18 nucleotides upstream from a 10 nucleotide oligo(A) tract. The coding region for b-IRBP would be expected to be 3300 bp long, but Northern blot hybridization experiments performed with bovine retina polyadenylated RNA and probes containing part of the coding region established that the mRNA for b-IRBP consisted of a major species of about 6300 bp, and a minor species of 5200 bp. In vitro translation of bovine retina polyadenylated RNA in a rabbit reticulocyte lysate system yielded an immunoreactive protein that was comparable in size with nonglycosylated, mature IRBP, showing that it is not synthesized from a large precursor, and supporting our finding that the mRNA contains an extensive non-coding region.

Cs2S04 gradients containing both Hg2+ and Ag+ effect the complete separation of satellite deoxyribonucleic acids having identical densities in neutral CsCl gradients.

A combination of both Ag(+) and Hg(2+) in Cs(2)SO(4) effects the complete separation of two DNAs having identical densities in CsCl. Satellite DNAs of hermit crab, Pagurus pollicaris, and lobster, Homarus americanus, have been isolated by this means.

Lentigo maligna and malignant melanoma.

Lentigo maligna, a precancerous lesion, is a brown-black irregularly pigmented freckle, usually occurring on the face of the elderly subject. In a series of 99 patients with malignant melanomas, lentigo maligna was the pre-existing lesion in 21. The clinical and histological findings, and previous publications on the subject are reviewed. Lentigo maligna itself is not a superficial malignant melanoma. After the development of malignant melanoma from lentigo maligna, eight of 21 patients developed metastatic disease. This seems to indicate that once malignant melanoma has developed (whether de novo from the junctional portion of a pre-existing nevus, or from a lentigo maligna), the outlook is the same. During the development of malignant melanoma from lentigo maligna there is an indefinite period when it is virtually impossible to determine histologically whether malignant melanoma is present. Naturally, the inclusion of these indefinite cases will greatly influence reported results of treatment.

Pediococci residing on plants.

The pediococci residing on plants resemble the lactobacilli, but they differ from the streptococci in their limited distribution and low population level on plants. They are a subgroup within the genus Pediococcus which grow freely in neutral media and require neither NaCl nor CO(2). They are most readily recognized by the ability to initiate growth in liquid media, acidified to pH 5.0, which contain 1.5% sodium acetate. In stained preparations the cells occur singly and in pairs, short chains, and clusters. The occurrence of two-dimensional tetrads may be rare; this varies with the individual culture and with the culture medium. The terminal pH in 2% glucose broth varies from 3.6 to 4.3. Ability to initiate growth at 45 C, production of ammonia from arginine, dissimilation of malate, and fermentation of arabinose are confirmatory characteristics. The subgroup contains only two quite similar, but differentiable, species. P. acidilactici initiates growth at 50 C and produces catalase on heated blood medium but does not produce acid-sensitive catalase; a majority of the strains fail to initiate growth at 10 C and many fail to ferment maltose and lactose. P. pentosaceus initiates growth at 10 C but not at 50 C and produces acid-sensitive catalase; catalase production on heated blood medium is transient; a majority of the cultures ferment maltose, salicin, and trehalose. No carbohydrate serves reliably to differentiate between the species. The guanine plus cytosine ratio of P. pentosaceus deoxyribonucleic acid (DNA) was determined to be 35.1 +/- 1.2 and that of P. acidilactici DNA is 38.5 +/- 0.8.

Multiple control elements for the uvrC gene unit of Escherichia coli.

We have sequenced the control region of the uvrC protein including two open reading frames (ORF) encoding polypeptides of 28 kd and 23 kd molecular weight. The uvrC gene is preceded by five promoters. The P1, P2a and P2b promoter sequences are 5' to the 28 kd and the 23 kd proteins respectively. The P3 and P4 promoters are located within the structural gene for the 23 kd protein. The P3 promoter is required for adequate in vivo expression. There are three putative lexA protein binding sites, detected at the 3' end of the 28 kd protein (lexA1), within the coding sequences for the 23 kd protein (lexA2) and within the P3 promoter (lexA3). Promoter P2 is responsible for transcription of the uvrC gene, producing transcripts of 2.8 and 1.6 kb. The upstream region including the 28 kd protein is required for enhanced expression under non-induced conditions. These results show that the uvrC gene is controlled by multiple promoters and is transcribed as part of a multigene unit.

Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc.

We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.

Advances in genosensor research.

Microfabricated devices containing arrays of nucleic acid hybridization sites, known as genosensors, are being developed for a variety of uses in genomic analysis. A great deal of the overall genosensor development effort involves optimization of experimental conditions in the actual use of genosensors. Here we describe a "low-tech" form of genosensor technology, involving arrays of oligonucleotides on glass microscope slides, which can be used to define optimal operating conditions and to develop applications of hybridization arrays in genome mapping and sequencing. In addition, we describe a porous silicon genosensor, which can be operated in a flowthrough mode, and discuss its advantages over current flat-surface designs. Porous silicon genosensors containing arrays of DNA fragments offer several unique capabilities in genome analysis.