Organization of chicken DNA sequences homologous to the transforming gene of avian myeloblastosis virus. I. Restriction enzyme analysis of total DNA from normal and leukemic cells.

Hybridization probes consisting of cloned DNA recombinants which represent different regions of the leukemogenic sequence (amv) from avian myeloblastosis virus were used to carry out a more detailed restriction endonuclease analysis of the homologous sequences (proto-amv) present in normal and leukemic chicken DNA. The results show that four large introns interrupt the normal cellular proto-amv sequences and that there is no major rearrangement of these sequences in leukemic myeloblasts.     

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.     

The smaller human VH gene families display remarkably little polymorphism.

We report the nucleotide sequence of 30 distinct human VH gene segments from the VHIV, VHV and VHVI gene families. When these sequences were compared to previously published sequences from these smaller human VH families a surprisingly low level of polymorphism was noted. Two VHIV gene segments from unrelated individuals were identical to two previously published VHIV sequences. Five VHV sequences were identical and seven VHVI gene segments were identical. Where differences were found between the sequences, allele specific oligonucleotide probes were used to verify the germline nature of the change and to test for segregation in several large kindreds. These data provide evidence that at least some human VH gene segments are remarkably stable.     

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspered tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment. The largest fragments would be those that contained one or more long minisatellite tracts. Some of these large fragments would be highly polymorphic by virtue of the included minisatellite sequences; by virtue of their complex structure, all would be capable of hybridizing to a wide variety of probes, uncovering a DNA fingerprint pattern.     

An Anchored Framework BAC Map of Mouse Chromosome 11 Assembled Using Multiplex Oligonucleotide Hybridization

Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an “overgo” computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.     

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.     

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.     

Chromosomal distribution of the RTVL-H family of human endogenous retrovirus-like sequences

We have analyzed the chromosomal distribution of a large family of human endogenous retrovirus-like sequences termed RTVL-H. In situ hybridizations suggest that these sequences are found on all human chromosomes. These results also indicate that clusters or concentrations of RTVL-H elements may exist on chromosomes 1p and 7q. Southern blotting experiments using somatic cell hybrids containing either the human chromosome 3 or the X chromosome confirm the presence of multiple dispersed RTVL-H sequences on these two chromosomes. These experiments also demonstrate that distinct RTVL-H banding patterns can be detected for each chromosome. Thus, RTVL-H probes may be useful in genome mapping studies.     

Temporal Expression of Mouse Glial Fibrillary Acidic Protein mRNA Studied by a Rapid In Situ Hybridization Procedure

A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week.     

Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect

Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C_ot-1 DNA). Contrary to expectation, we demonstrated that C_ot-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C_ot-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C_ot-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using C_ot-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of C_ot-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for C_ot-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.     

Number and organization of actin-related sequences in the mouse genome

Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.     

Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray

Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all possible mismatch probes is impractical. Our study provides guidelines for designing non-perfectly matched DNA probes to target DNA sequences as desired throughout the genome. These guidelines are based on the analysis of hybridization data between perfectly matched and non-perfectly matched DNA sequences (single-point or double-point mutated) calculated in silico. Large changes in hybridization temperature predicted by these guidelines for non-matched oligonucleotides fit independent experimental data very well. Applying the guidelines to find oligonucleotide microarray probes for P450 genes, we confirmed the ability of our point mutation method to differentiate the individual genes in terms of thermodynamic calculations of hybridization and sequence similarity.     

Isolation and Characterization of New Maize B Sequences from a Microdissected Library

Understanding the evolution of the maize B chromosome requires insight into the molecular organization of a large number of B clones, which can be potentially obtained by microdissection of the chromosome. Yet, the microdissection protocols currently available are ineffective for a large-scale isolation. In an attempt to improve its efficiency, a protocol was adopted to screen a microdissected B library with probes prepared from the degenerate oligonucleotide primed-PCR product of genomic DNA. This protocol resulted in 59 new B clones, most of which were highly repetitive sequences located in various B regions but mostly in the heterochromatic blocks of the long arm. They also appeared in A chromosomes. Twenty-four of these were retrotransposons, ten knob, 18 noncoding sequences, and seven unknown sequences. The implication of the new B sequences on the B evolution is discussed.     

Comparison of class I (H-2) gene sequences. Derivation of unique probes for members of this multigene family

The genes for the classical transplantation antigens are unique in that they belong to a multigene family of which each member is represented by a large number of alleles. Since all of these genes are highly related in sequence, it has been difficult to study the expression of individual members of this complex gene family. Based upon our initial suggestion that the 3' noncoding regions of these genes may be useful in identifying mRNA molecules transcribed from different loci, we have compared a large number of sequences from different inbred mouse strains and have been able to assign each of these sequences without ambiguity into distinct allelic series. Such accurate assignment has afforded the opportunity to compare the coding regions of these highly homologous genes and has led to the identification of sequences which are apparently unique to specific genes in the family. Synthetic oligonucleotides corresponding to each of the locus-specific unique regions have been used successfully to type a panel of cDNA sequences, as well as to quantitate the relative amounts of mRNA transcribed from distinct loci. The availability of these specific coding probes will allow the analysis of individual genes and their specific expression without interference from other highly homologous sequences in this multigene family.     

K-tuple frequency in the human genome and polymerase chain reaction.

The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.     

In silico evaluation of molecular probes for detection and identification of Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus

Ralstonia solanacerum and Clavibacter michiganensis subsp. sepedonicus are the two most relevant bacterial pathogens of potato for which a large number of molecular diagnostic methods using specific DNA sequences have been developed. About one hundred oligonucleotides have been described and thoroughly tested experimentally. After having compiled and evaluated all these primers and probes in silico to check their specificity, many discrepancies were found. A detailed analysis permitted the recognition of different possible reasons for such discrepancies: sequencing errors in public sequences, wrong supposed specificity (sometimes due to more recent sequences than the oligonucleotides being evaluated) or even typing errors in the oligonucleotides. Although this study is an exercise about in silico evaluation using two potato bacterial pathogens as a model, the conclusions reflect not only information useful for phytopathologists but, in a broader scope, draw the main situations that can be found during an evaluation of probes, which can be surely found in other scenarios.     

New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA

Nucleic acid-templated chemistry is a promising strategy for imaging genetic sequences in living cells. Here we describe the synthesis of two new nucleophiles for use in templated nucleophilic displacements with DNA probes. The nucleophilic groups are phosphorodithioate and phosphorotrithioate; we report on synthetic methods for introducing these groups at the 3'-terminus of oligonucleotides. Both new nucleophiles are found to be more highly reactive than earlier phosphoromonothioates. This increased nucleophilicity is shown to result in more rapid templated reactions with electrophilic DNA probes. The new probes were demonstrated in detection of specific genetic sequences in solution, with clear signal over background being generated in as little as 20 min. The probes were also tested for imaging ribosomal RNA sequences in live Escherichia coli; useful signal was generated in 20 min to 1h, approximately one quarter to one-half the time of earlier monothioate probes, and the signal-to-noise ratio was increased as well.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.