Hybridization Of Restriction Fragments Research Articles

Quantitative Genome-Wide Measurements of Meiotic DNA Double-Strand Breaks and Protein Binding in S. pombe.

The fission yeast Schizosaccharomyces pombe is especially well suited for studying meiosis in molecular detail. Experiments with S. pombe strains that undergo a nearly synchronous meiosis-at variable temperatures-have elucidated the mechanisms of meiotic progression and the proteins that are involved. For example, studies focused on the initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs) have proven exceptionally informative. In meiosis, some regions of DNA have more frequent DSBs than the surrounding regions. These DSB hotspots can be visualized by Southern blot hybridization of restriction fragments ranging from kilobases (kb) to megabases (Mb) in size. More recently, the benefits of genome-wide analysis to map the distribution and frequency of meiotic DSBs have been attained, with resolution down to the nucleotide level. Infrequent, non-hotspot DSBs previously not detectable have been observed, creating a better understanding of how recombination is regulated. Additional genome-wide analyses have shown proteins that bind specifically to DSB hotspots, providing insight into how the DSB initiation complex functions. We describe here detailed methods for achieving these results.

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

Efficient Tracing of Global Isolates of Yersinia pestis by Restriction Fragment Length Polymorphism Analysis Using Three Insertion Sequences as Probes

Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from rodent to rodent and from rodent to humans by fleabites. Multiple copies of three insertion sequences (IS100, IS285, and IS1541) are scattered over the Y. pestis genome. The genomic instability generated by these insertion sequences (IS) creates a polymorphism of the hybridizing restriction fragments (restriction fragment length polymorphism [RFLP]) which can be used to subtype this relatively clonal species. The aim of this work was to evaluate and compare the potential of the three IS-RFLP techniques, individually or in combination, to define clusters of strains according to their focus of origin. The analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each IS-RFLP used individually. In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination (D = 0.999) and a robust clustering of the isolates according to their biovar and geographical origin. This geographical clustering was observed even within the Orientalis group, although these strains had only a short period of time (one century) to diverge from the original clone that spread globally. Therefore, 3IS-RFLP is a technique that may be useful for addressing epidemiological problems and forensic issues. When plague reemerges after several decades of silence in a quiescent focus, it may help in determining whether the disease was reimported or reactivated. It may also be of value to identify the origin of a strain when plague cases appear in a previously plague-free region. Finally, this technique could be useful for the tracing of a Y. pestis isolate that has been used as a biological terrorism threat.

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.

Genetic Localization of a Drosophila melanogaster Resistance Gene to a Parasitoid Wasp and Physical Mapping of the Region

Drosophila melanogaster larvae usually react against eggs of the parasitoid wasp Leptopilina boulardi by surrounding them with a multicellular melanotic capsule. The genetic determinism of this response has been studied previously using susceptible (non-capsule-forming) and resistant (capsule-forming) strains. The results suggest that differences in their encapsulation response involve a single gene, resistance to Leptopilina boulardi (Rlb), with two alleles, the resistant one being dominant. Rlb confers specific protection against Leptopilina boulardi and is thus probably involved in parasitoid recognition. Recent studies have localized this gene on the right arm of the second chromosome and our aim was to precisely determine its genetic and molecular location. Using strains bearing deletions, we demonstrated that resistance to Leptopilina boulardi is conferred by the 55C; 55F3 region and that the 55E2-E6; F3 region is particularly involved. A physical map of the 55C; 56A region was then constructed, based on a set of overlapping cosmid and P1 phage clones. Using single and double digests, cross hybridization of restriction fragments, and location of genetically mapped genes and STSs, a complete, five-enzyme restriction map of this 830-kb region was obtained.

A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus x domestica by Venturia inaequalis infection and salicylic acid treatment.

A cDNA clone encoding a leucine-rich repeat (LRR) receptor-like protein kinase (LRPKm1) of Malus x domestica cv. Florina has been isolated using as a heterologous probe a cloned gene encoding a polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. A genomic clone containing the 5'-regulatory region and a 5' portion of the open reading frame of the LRPKm1 gene has also been isolated. An open reading frame of 2997 nt (999 amino acids) was present in the cDNA clone, encoding a receptor-like protein comprising a 21 amino acid signal peptide for secretion, a leucine zipper, 23 LRRs, a putative membrane-spanning region and a serine/threonine protein kinase domain. LRPKm1 shows homology to the A. thaliana receptor-like protein kinase RLK5 and, to a minor extent, to PGIP. The LRPKm1 region from +5 to +600 exhibits an alternative reading frame that encodes a product corresponding to a proline-rich protein fragment homologous to several hydroxyproline-rich proteins. Southern blot analysis showed that LRPKm1 belongs to a multigene family and that there is length polymorphism of the hybridizing restriction fragments among different M. x domestica cultivars. Northern blot analysis was carried out on mRNA extracted from infected leaves of either cv. Florina (resistant to Venturia inaequalis) or cv. Golden Delicious (susceptible to V. inaequalis), and from tissues treated with salicylic acid. A 3500 bp transcript hybridizing at high stringency with the LRPKm1 cDNA accumulated in response to infection or salicylic acid treatment. Transcript accumulation was more intense in the incompatible interaction than in the compatible one. The possible involvement of this receptor-like protein kinase in resistance of apple to phytopathogenic fungi is discussed.

Telomerase activity in melanoma and non-melanoma skin cancer.

Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found. © 1999 Cancer Research Campaign

Seed-storage-protein loci in RFLP maps of diploid, tetraploid, and hexaploid wheat

Linkages between high- and low-molecular-weight (M(r)) glutenin, gliadin and triticin loci in diploid, tetraploid and hexaploid wheats were studied by hybridization of restriction fragments with DNA clones and by SDS-PAGE. In tetraploid and hexaploid wheat, DNA fragments hybridizing with a low-M(r) glutenin clone were mapped at the XGlu-3 locus in the distal region of the maps of chromosome arms 1AS, 1BS, and 1DS. A second locus, designated XGlu-B2, was detected in the middle of the map of chromosome arm 1BS completely linked to the XGli-B3 gliadin locus. The restriction fragments mapped at this locus were shown to co-segregate with B subunits of low-M(r) glutenins in SDS-PAGE in tetraploid wheat, indicating that X Glu-B2 is an active low-M(r) glutenin locus. A new locus hybridizing with the low-M(r) clone was mapped on the long arm of chromosome 7A(m) in diploid wheat. No glutenin protein was found to co-segregate with this new locus. Triticin loci were mapped on chromosome arms 1AS, 1BS, and 1DS. A failure to detect triticin proteins co-segregating with DNA fragments mapped at XTri-B1 locus suggests that this locus is not active. No evidence was found for the existence of Gli-A4, and it is concluded that this locus is probably synonymous with Gli-A3. Recombination was observed within the multigene gliadin family mapped at XGli-A1 (1.2 cM). Although these closely linked loci may correspond to the previously named Gli-A1 and Gli-A5 loci, they were temporarily designated X Gli-A1.1 and X Gli-A1.2 until orthology with Gli-A1 and Gli-A5 is established.

Construction of a 1-Mb Restriction-Mapped Cosmid Contig Containing the Candidate Region for the Familial Mediterranean Fever Locus (MEFV) on Chromosome 16p13.3

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning ∼1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the ∼700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported.NotI andEcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor.

A total of four cytomegalovirus (CMV) isolates were obtained from two CMV seronegative patients, each of whom received a lung transplant from the same seropositive donor. CMV was isolated from Patient 1 from two bronchial alveolar lavage (BAL) specimens, one obtained during treatment with ganciclovir (GCV) and a second during later treatment with foscarnet. Both of these isolates are sensitive to GCV and foscarnet. CMV was isolated from Patient 2 from a blood and a BAL specimen obtained during treatment with GCV. Both of these isolates are resistant to GCV and show reduced GCV phosphorylation. Patient 1 is still alive 33 months posttransplant. Patient 2 died 6 1/2 months posttransplant. Although the four strains differ with respect to GCV susceptibility and phosphorylation, their DNA restriction fragment hybridization patterns and UL97 kinase gene sequences indicate that they are closely related. The restriction fragment hybridization patterns are identical among the strains, while these patterns differ markedly from those of unrelated strains. The DNA sequences of the UL97 genes of the strains from Patient 2 differ by only one nucleotide from those of Patient 1. The same comparison with unrelated strains shows a minimum of 12 nucleotide differences. The nucleotide change in the strains from Patient 2 produces an amino acid substitution of serine for leucine at residue 595, a substitution that was previously shown to transfer GCV resistance. Both patients, therefore, were apparently infected with the same donor strain, but during the course of GCV prophylaxis and treatment, a GCV-resistant mutant strain was selected in Patient 2.

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.

Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.

Toxic shock syndrome toxin 1-producing Staphylococcus aureus isolates contain the staphylococcal enterotoxin B genetic element but do not express staphylococcal enterotoxin B.

Toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin B (SEB) are two examples of variable genetic traits produced by Staphylococcus aureus. Regarding phenotypes, most investigations have reported that SEB and TSST-1 are not coproduced in S. aureus. However, it is unclear whether genotypically SEB (entB) and TSST-1 (tst) genes are found within the same isolate. Gene probes specific for entB and tst showed that both genes were present within 8 TSST-1+ isolates analyzed. However, SEB was not produced by any of these isolates. In contrast, none of 9 SEB-producing S. aureus isolates contained the TSST-1 genetic element DNA. Hybridization of restriction fragments with TSST-1 and SEB genetic element probes and tryptophan auxotyping suggested that tst and entB are located in close proximity on the chromosome. The entB locus may be a preferred site of insertion for the TSST-1 genetic element, and such insertion may interfere with the expression of SEB.

Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus Sclerotinia sclerotiorum

The phytopathogenic fungus Sclerotinia sclerotiorum produces a number of extra-cellular pectin-degrading enzymes. We have cloned and determined the complete sequence of a gene ( pgl) encoding an endopolygalacturonase (PG1). The coding region consists of a non-interrupted 1143-bp open reading frame. S. sclerotiorum pg1 was compared to other fungal PG-encoding genes. Basic transcription control sequences were identified in the 5' non-coding region.The deduced amino acid (aa) sequence (380 aa) of the enzyme is compared to seven fungal PG sequences and shows a high level of identity (41.5 to 59.8%). Predicted secondary structures were compared, revealing a similar protein organization most probably in antiparallel β sheets. Hybridization analysis using a pg1 0.65-kb BamHI fragment as a probe allowed the identification of seven different recombinant phages from a genomic library. Analysis of the hybridizing restriction fragments suggests that PG-encoding genes are organized as a family.

The genomic organization of the rat angiotensin II receptor AT 1B

Expression of two subtypes of angiotensin type 1 receptor (AT 1A and AT 1B) seems to be controlled by different mechanisms. As a step toward the elucidation of difference in the mechanism in their gene expression, the genomic organization of rat angiotensin II type 1B receptor was investigated. Rat genomic DNA fragments were cloned by screening a genomic library with fragments from two cDNA clones (2.3 and 2.2 kb) encoding the rat AT 1B receptor. Three λ clones were isolated, and the hybridizing restriction fragments were sequenced. Comparison of the genomic clone DNA and cDNA sequences revealed that the rat AT 1B receptor gene contains three exons and two introns. Two of the exons encoded 5′ untranslated sequences while the third exon contained the entire coding region. The 5′-flanking region contained the typical sequence motifs found in many eukaryotic promoters including a TATA box and a potential SP1 binding site. The transcription initiation site was located at −8 bp upstream the 5′ end of the cDNA in the 5′-flanking region.

Molecular characterization of a recurring complex chromosomal translocation in two human extragonadal germ cell tumors

The molecular characterization of a recurring complex chromosomal translocation involving 6p21, 6p22, 6q23, and 11q13 in two independent but similar extragonadal human germ cell tumors was initiated using fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) techniques. By using a series of specific probes from the 11q13 region, the translocation breakpoint in this chromosomal band could be located within a long-range restriction enzyme map in between the markers D11S457 and D11S546. In addition, aberrantly hybridizing restriction fragments were revealed by PFGE in both tumors, indicating that the breakpoint region must be located within a distance of at maximum 200 kilobase pairs (kbp) from the nearest DNA marker (D11S546).

Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis

The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups.

Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.

Cytological localization of thePGIP genes in the embryo suspensor cells ofPhaseolus vulgavis L

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein which inhibits fungalendopolygalacturonases. A small gene family encodesPGIP in the genome of common bean, as indicated by Southernblot experiments performed at high-stringency conditions. Southern-blot analysis of DNA extracted from different cultivars ofPhaseolus vulgaris and fromPhaseolus coccineus showed length polymorphism of the hybridizing restriction fragments. The cytological localization of thePGIP genes was determined in polytene chromosomes of theP. vulgaris embryo suspensor cells. In-situ hybridization experiments using the clonedPGIP gene revealed labelling over a single region of the pericentromeric heterochromatin of chromosome pair X, next to the euchromatin, suggesting thatPGIP gene family may be clustered in one chromosomal region.

Hybridization profiles and restriction fragment length polymorphisms for bovine and ovine interferon genes

Abstract Bovine α‐ (IFNA), β‐ (IFNB), ω‐ (IFNW), trophoblast (IFNT), and γ‐(IFNG) interferon (IFN) cDNA probes were hybridized to bovine and ovine DNA samples digested with a panel of 7 restriction enzymes to characterize the hybridization patterns and genetic variability of IFN genes in these two species. Sixty percent of probe‐enzyme combinations identified RFLPs in cattle compared to 46% of the combinations in sheep. The presence of polymorphic bands in each probe‐enzyme combination was compared between species. Bovine IFNA and IFNW RFLPs were observed in cattle with all 7 enzymes while ovine IFNA and IFNW RFLPs were found with 5 and 2 enzymes, respectively. IFNG RFLPS were observed with HinD III (61% of cattle screened), Taq I (28% of sheep) and Pst I (56% of sheep). IFN hybridization profiles were more complex in sheep, but RFLPs were more common in cattle. The most useful enzyme to screen for IFN RFLPs was found to be HinD III in cattle and Taq I in sheep.