DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA.

Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two-step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full-length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates.

Interdependence between DNA template secondary structure and priming efficiencies of short primers.

Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.

DNA sequencing using differential extension with nucleotide subsets (DENS).

Here we describe template directed enzymatic synthesis of unique primers, avoiding the chemical synthesis step in primer walking. We have termed this conceptually new technique DENS (differential extension with nucleotide subsets). DENS works by selectively extending a short primer, making it a long one at the intended site only. The procedure starts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs. The primer is extended by 6-9 bases or longer at the intended priming site, which is deliberately selected, (as is the two-dNTP set), to maximize the extension length. The subsequent termination reaction at 60-65 degrees C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. DENS allows the use of primers as long as 8mers (degenerate in two positions) which prime much more strongly than modular primers involving 5-7mers and which (unlike the latter) can be used with thermostable polymerases, thus allowing cycle-sequencing with dye-terminators compatible with Taq DNA polymerase, as well as making double-stranded DNA sequencing more robust.

Ubiquitous expression of a cloned murine thymopoietin cDNA.

Thymopoietin (TP) was originally isolated as a 5-kD 49-aa protein from bovine thymus and was subsequently observed to affect T-cell differentiation and function. We report here the molecular cloning of a murine TP cDNA. The 2,514 bp fragment contains a 630 bp open reading frame that encodes for 210 aa, highly homologous to the first 220 aa of the human TP beta and TP gamma isoforms and to bovine TP. Southern blot analysis of genomic DNA revealed that in mouse, calf and human TP is encoded by a single genomic locus. Recently, it was found that one of the TP isoforms designated TP beta is a homologous protein to the lamina-associated polypeptide 2 (LAP2), which is thought to play an important role in the regulation of nuclear architecture by binding lamin B1 and chromosomes in a manner regulated by phosphorylation during mitosis. In this study we report the TP expression at the transcription level in 17 murine and rat tissues of different origins and 18 lymphoid and nonlymphoid cell lines. The assessment of TP mRNA expression by S1-nuclease protection assays and in situ hybridizations revealed that its expression is not exclusive to thymus, but rather ubiquitous, higher in lymphatic tissues, but also in other cells characterized with a high rate of proliferation. TP was also shown to be expressed in athymic and old animals, lacking a functional thymus gland. Further in situ hybridization studies revealed that within the thymus, the highest levels of TP mRNA are noted at the cortex. These results suggest a possible role for TP in proliferation and cell cycle control.

On the mechanism of the modular primer effect.

Modular primers are strings of three contiguously annealed unligated oligonucleotides (modules) as short as 5- or 6-mers, selected from a presynthesized library. It was previously found that such strings can prime DNA sequencing reactions specifically, thus eliminating the need for the primer synthesis step in DNA sequencing by primer walking. It has remained largely a mystery why modular primers prime uniquely, while a single module, used alone in the same conditions, often shows alternative priming of comparable strength. In a puzzling way, the single module, even in a large excess over the template, no longer primes at the alternative sites, when modules with which it can form a contiguous string are also present. Here we describe experiments indicating that this phenomenon cannot be explained by cooperative annealing of the modules to the template. Instead, the mechanism seems to involve competition between different primers for the available polymerase. In this competition, the polymerase is preferentially engaged by longer primers, whether modular or conventional, at the expense of shorter primers, even though the latter can otherwise prime with similar or occasionally higher efficiency.

DNA sequencing: modular primers assembled from a library of hexamers or pentamers.

Here we report a striking effect displayed by "modular primers," which consist of hexamer or pentamer oligonucleotide modules base-stacked to each other upon annealing to a DNA template. Such a combination of modules is found to prime DNA sequencing reactions uniquely, unlike either of the modules alone. We attribute this effect in part to the increase in the affinity of an oligonucleotide for the template in the presence of an adjacent module. All possible pentamer (or hexamer) sequences total 1024 (or 4096) samples, a manageable size for a presynthesized library. This approach can replace the synthesis of primers, which is the current bottleneck in time and cost of the primer walking sequencing, and can allow full automation of the closed cycle of walking.

Molecular cloning of the bovine thymopoietin gene and its expression in different calf tissues: evidence for a predominant expression in thymocytes.

Thymopoietin (TP), a 49 amino acid peptide, is regarded as a thymic hormone, secreted specifically by some epithelial cells in the thymic stroma and exerting a multitude of effects on maturation and function of T lineage cells. As part of our study on the molecular biology of TP, we isolated cDNA clone coding for a bovine TP precursor and used it as a probe to analyze the presence of mRNA coding for TP in different tissues. The cDNA clone reported here is 1.1 kb long and contains an open reading frame (ORF) of 741 bp which corresponds to 247 amino acids. The 147 bp coding for the entire bovine TP are at the 5' end of the ORF. A DNA fragment coding for amino acids 1-42 of bovine TP was used as a probe to look for hybridizable RNA sequences, extracted from various calf tissues, by the S1 nuclease protection method. Our results indicate that the TP gene is expressed predominantly in lymphatic tissues. Lymphatic tissues with the highest levels observed were thymocytes and not thymic stroma. Lower, but still significant, amounts were present in tonsils, neck lymph nodes, and small intestine (probably because of its lymphatic part--the Peyer's patches), whereas cultured thymic stromal cells, spleen tissue and peripheral blood mononuclear cells displayed a low level of TP mRNA. The TP gene expression in all other (non-lymphatic) tissues tested, was weak, barely detectable or virtually absent. However, the cerebellum could be singled out with relatively strong expression of TP mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.

Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.

Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides.

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16.

A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16p11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues.

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.

The use of mRNA translation in vitro and in ovo followed by crossed immunoelectrophoretic autoradiography to study the biosynthesis of human cholinesterases.

The synthesis of various cholinesterases in different fetal human tissues was studied using in vitro and in ovo translation of poly(A)+ RNA, followed by crossed immunoelectrophoretic autoradiography. When unfractionated poly(A)+ mRNA from fetal brain, muscle, or liver was translated in vitro, in the reticulocyte lysate cell-free system, polypeptides were synthesized which reacted with antibodies against either "true" acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or "pseudo", butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8). The two nascent cholinesterases could be separated by crossed immunoelectrophoresis followed by autoradiography, suggesting that acetylcholinesterase and butyrylcholinesterase are produced in all three tissues from nascent polypeptides containing different immunological domains. To examine whether the biosynthesis of cholinesterases includes posttranslational processing events, Xenopus oocytes were microinjected with mRNA from these tissues. Immunoelectrophoretic analysis of oocyte intracellular homogenates and incubation medium revealed various precipitation arcs, reflecting the synthesis and posttranslational processing of multiple forms of tissue-specific exported and intracellular acetylcholinesterase and butyrylcholinesterase. These findings demonstrate that polymorphic cholinesterases are produced from nascent polypeptide products which undergo further posttranslational processing events in a tissue-specific manner before they become mature compartmentalized cholinesterases.

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue.

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.

Use of synthetic oligodeoxynucleotide probes for the isolation of a human cholinesterase cDNA clone.

Cholinesterases are serine esterases that rapidly hydrolyze the neurotransmitter acetylcholine. In humans, cholinesterases exhibit extensive polymorphism in terms of their substrate specificity, sensitivity to selective inhibitors, hydrophobicity, and cellular as well as subcellular localization. It is not yet known whether the various cholinesterase forms originate from different genes or are products of posttranscriptional and posttranslational processing. The extent to which these enzyme forms are homologous in their amino acid sequence is also not known. However, a consensus organophosphate-binding hexapeptide sequence Phe-Gly-Glu-Ser-Ala-Gly was found both in "true" acetylcholinesterase from the electric organ of Torpedo [McPhee-Quigley et al: J Biol Chem 260:12185-12189, 1985] and in "pseudocholinesterase" (butyrylcholinesterase) from human serum [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter pp 5-12, 1984], suggesting that this region in the protein is conserved in all cholinesterases. Based on this common sequence, we prepared synthetic oligodeoxynucleotides and used them as labeled probes to screen a cDNA library from fetal human brain mRNA, cloned in lambda gt10 phages. A cDNA clone of 770 nucleotides in length was isolated. It contains an open reading frame terminating with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala, which includes the consensus hexapeptide used for designing the DNA probe. Furthermore, the sequence of this 12-amino acid peptide is identical to the sequence reported for the organophosphate binding site of human serum pseudocholinesterase [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter, pp 5-12, 1984]. These findings confirm that the isolated clone is indeed part of a human cholinesterase cDNA.

A human acetylcholinesterase gene identified by homology to the Ace region of Drosophila.

The Ace locus of the Drosophila genome controls biosynthesis of the neurotransmitter-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). We injected the mRNA species hybridizing with DNA fragments from this region into Xenopus oocytes, in which acetylcholinesterase mRNA is translated into active acetylcholinesterase. A 2.0-kilobase (kb) fragment of DNA from this region selectively hybridizes with Drosophila mRNA capable of inducing the biosynthesis of acetylcholinesterase in oocytes. This Drosophila DNA fragment cross-hybridized with human brain poly(A)+ RNA. We therefore used this DNA fragment as a probe for homologous sequence(s) in a human genomic DNA library and thus selected a 13.5-kb human DNA segment. DNA blot-hybridization revealed that a 2.6-kb fragment of this human DNA segment hybridizes with the Drosophila 2.0-kb DNA fragment. Both Drosophila and human fragments hybridized with a human brain mRNA species of about 7.0-kb that was barely detectable in the acetylcholinesterase-deficient HEp carcinoma. A fraction containing mRNA of similar size, extracted from human brain, induced acetylcholinesterase biosynthesis in oocytes. The human DNA fragment also was used in hybridization-selection experiments. In oocytes, hybrid-selected human brain mRNA induced acetylcholinesterase activity that was completely inhibited by 1,5-bis[4-allyldimethylammonium)phenyl]pentan-3-one dibromide but not by tetraisopropyl pyrophosphamide, a differential response to these inhibitors characteristic of "true" human brain acetylcholinesterase. These findings strongly suggest that both the Drosophila and the human DNA fragments are directly involved in controlling acetylcholinesterase biosynthesis.

Expression of acetylcholinesterase gene(s) in the human brain: molecular cloning evidence for cross-homologous sequences.

The regulation of acetylcholinesterase (AChE) in the human brain has been approached at the level of the genome. A human DNA fragment of the length of 2 600 nucleotides was isolated from a human genomic library. This DNA fragment, designated Huache 1R, bears sequence homology to a DNA fragment from the vicinity of the Drosophila Ace region, that controls AChE biosynthesis (Soreq et al., 1985). Polyadenylated RNA from human brain was hybridized with Huache 1R DNA, eluted and microinjected into Xenopus oocytes in the absence or presence of 35S-methionine. The hybrid-selected RNA induced the biosynthesis of active AChE in the oocytes. Immunoprecipitation of labeled oocyte proteins with monoclonal antibodies against human AChE (Fambrough et al., 1982) resulted in the selective precipitation of an 85 000 Mr induced protein, with a similar size to that of the subunit of human brain AChE. These findings show that the Huache 1R DNA hybridizes with human brain AChEmRNA. The Huache 1R fragment was employed to select a collection of 12 homologous phage-cloned human genomic DNA fragments with different restriction patterns. A cDNA library in pBR322 plasmids was prepared from polyadenylated RNA isolated from embryonic brain. This library was also screened using labeled Huache 1R DNA as a probe. Forty-two out of 37 000 colonies were found positive. Several of these were selected for further analyses. Hybrid-selection experiments using DNA from two of the positive plasmid clones showed that these cDNAs also hybridize with AChEmRNA from human brain. DNA blot hybridization revealed homologies between these cDNA chains and the original Huache 1 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.

Skeletal muscle actin mRNA. Characterization of the 3' untranslated region.

Plasmids p749, p106, and p150 contain cDNA inserts complementary to rat skeletal muscle actin mRNA. Nucleotide sequence analysis indicates the following sequence relationships: p749 specifies codons 171 to 360; p150 specifies codons 357 to 374 together with 120 nucleotides of the 3'-non-translated region; p106 specifies the last actin amino acid codon, the termination codon and the entire 3' non-translated region. Plasmid p749 hybridized with RNA extracted from rat skeletal muscle, cardiac muscle, smooth (stomach) muscle, and from brain. It also hybridizes well with RNA extracted from skeletal muscle and brain of dog and chick. Plasmid p106 hybridized specifically with rat striated muscles (skeletal and cardiac muscle) mRNA but not with mRNA from rat stomach and from rat brain. It also hybridized to RNA extracted from skeletal muscle of rabbit and dog but not from chick. Thermal stability of the hybrids and sensitivity to S1 digestion also indicated substantial divergence between the 3' untranslated end of rat and dog skeletal muscle actins. The investigation shows that the coding regions of actin genes are highly conserved, whereas the 3' non-coding regions diverged considerably during evolution. Probes constructed from the 3' non-coding regions of actin mRNAs can be used to identify the various actin mRNA and actin genes.