Isolation Of Several Clones Research Articles

Degenerate RNA Packaging Signals in the Genome of Satellite Tobacco Necrosis Virus: Implications for the Assembly of a T = 1 Capsid

Using a recombinant, T = 1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with “free” CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem–loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem–loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem–loops displaying the sequence motif AxxA. The implication is that there are many stem–loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem–loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA.

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.

The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.

Comparison of PCR-based mutation detection methods and application for identification of mouseSult1a1 mutant embryonic stem cell clones using pooled templates

Reverse genetic approaches to generate mutants of model species are useful tools to assess functions of unknown genes. Recent work has demonstrated the feasibility of such strategies in several organisms, exploiting the power of chemical mutagenesis to disrupt genes randomly throughout the genome. To increase the throughput of gene-driven mutant identification, efficient mutation screening protocols are needed. Given the availability of sequence information for large numbers of unknown genes in many species, mutation detection protocols are preferably based on PCR. Using a set of defined mutations in the Hprt1 gene of mouse embryonic stem (ES) cells, we have systematically compared several PCR-based point mutation and deletion detection methods available for their ability to identify lesions in pooled samples, which is a major criterion for an efficient large-scale mutation screening assay. Results indicate that point mutations are most effectively identified by heteroduplex cleavage using CEL I endonuclease. Small deletions can most effectively be detected employing the recently described "poison" primer PCR technique. Further, we employed the CEL I assay followed by conventional agarose gel electrophoresis analysis for screening a library of chemically mutagenized ES cell clones. This resulted in the isolation of several clones harboring mutations in the mouse Sult1a1 locus, demonstrating the high-throughput compatibility of this approach using simple and inexpensive laboratory equipment.

Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene.

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5'UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5'upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3' end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5'UTR, similar to that described in the rat oxytocin receptor gene. 5'RACE and RNase protection analysis mapped two major putative transcription start points at -830 and -861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP-1 and AP-2 sites and a cluster of Sp1 sites upstream of the AP-2 sites. A luciferase construct containing a 2.1-kb of putative promoter, and part of the 5'UTR including the first intron, showed promoter activity when transfected into COS-7, CHO and PC12 cell lines but not in AtT-20 cells. A similar construct without the intron and distal 5'UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5'flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.

Differential gene expression during Trypanosoma cruzi metacyclogenesis.

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

A plasmid vector for isolation of strong promoters in Escherichia coli.

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

A plasmid vector for isolation of strong promoters in Escherichia coli

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

Characterization of a beta-actin mRNA zipcode-binding protein.

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the beta-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37 degrees C and in Lee medium at 25 degrees C and during formation of germ tubes in Lee medium 37 degrees C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.

The Human Glutamate Dehydrogenase Gene Family: Gene Organization and Structural Characterization

Glutamate dehydrogenase is a mitochondrially located, key metabolic enzyme. In addition to its general metabolic role, GLUD is important in neurotransmission. Significant alterations in GLUD enzymatic activity have been associated with certain neurodegenerative human disorders. Although a single species of human GLUD cDNA molecule has been identified so far, both genomic DNA Southern and cytogenetic analyses have indicated the presence of a GLUD gene family. Screening of a human genomic λ-phage library with the GLUD cDNA, led us to the isolation of several clones divided into five structurally distinct contigs. We have confirmed the presence of all GLUD-specific sequences in the human genome by detailed genomic Southern analysis. This study allowed the identification of the entire functional GLUD gene, named GLUD1. The GLUD1 gene is about 45 kb long and it is organized into 13 exons. Its nucleotide sequence, exon-intron boundaries, and transcription start sites were determined. Potential binding sites for various regulatory factors such as Sp1, AP-1, and AP-2 were recognized at the promoter region of the gene. The members of the other contigs showed an organization clearly different from GLUD1 . Two distinct GLUD-specific gene loci, termed GLUDP2 and GLUDP3 , possibly represent truncated pseudogenes. Their high degree of similarity to GLUD1 is limited to the region surrounding exons 2, 3, and 4. Finally, two additional GLUD-specific genomic sequences, termed GLUDP4 and GLUDP5, are structurally similar with the 3′ part of the GLUD cDNA sequence. These loci probably represent truncated GLUD pseudogenes generated by retrotransposition. The data presented here suggest, that all human GLUD pseudogenes have diverged recently in evolution.

Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii

The monoclonal antibody (mAb) TG17.179 recognizes an excreted-secreted antigen (ESA) of 28.5 kDa named Gra 2, which is stored in the dense granules of Toxoplasma cells and secreted into the parasitophorous vacuole after host cell invasion. Screening of an expression cDNA library with TG17.179 led to the isolation of several clones, the longest one (clone L) being of 1030 bp. Clone L cDNA was found to be homologous to a previously described composite cDNA encoding a P28 protein of Toxoplasma gondii. Characterization of one genomic clone indicates that the complete GRA 2 gene is about 1.3 kb in length, including an intron of 241 bp. Northern blot and primer extension analyses confirmed the size of the mature messenger (1.1 kb). Amino acid partial sequencing of the native antigen purified by HPLC and metabolic radiolabelings of ESAs perfectly matched the primary amino acid structure deduced from the clone L cDNA. This primary translation product consists of an 185 amino acid polypeptide (19.8 kDa) including a 23 amino acid signal sequence. The presence of many serine and threonine residues may indicate an O-glycosylation. The predicted mature polypeptide shows an internal helical domain with 2 amphipathic α-helices. These might be involved in the association of Gra 2 with the membranous network within the parasitophorous vacuole.

Mitochondrial Cytochrome C Oxidase II Messenger Ribonucleic Acid is Expressed in Pachytene Spermatocytes at High Levels and in a Stage-Dependent Manner during Spermatogenesis in the Rat

Screening of a rat seminiferous tubule library (stages VI-VIII) resulted in isolation of several clones to the same abundant mRNA. Sequencing revealed that these encoded all or part of the second subunit of cytochrome C oxidase (COX II), the terminal enzyme in the electron transport chain located in mitochondria. The pattern of expression of the COX II mRNA in the testis was examined by Northern blot and in situ hybridisation combined with specific depletion of pachytene spermatocytes or Leydig cells by treatment with methoxyacetic acid (MAA) or ethane dimethane sulphonate (EDS), respectively. COX II mRNA was found to be strongly expressed in pachytene spermatocytes in adult and prepubertal rats, with the highest levels of expression at stages VII-VIII of the spermatogenic cycle. Although this is the androgen-dependent stage, Northern analysis of total testicular RNA showed that expression of COX II mRNA was not altered detectably by EDS-induced androgen withdrawal nor was expression altered detectably by 24-h treatment with high doses of FSH. Treatment of rats with MAA, which selectively depletes seminiferous tubules at most stages of pachytene spermatocytes, resulted in a marked reduction in COX II mRNA. In the absence of pachytene spermatocytes, COX II mRNA was visualized in preleptotene spermatocytes and in Sertoli cell cytoplasm. The expression of COX II in Sertoli cells was confirmed by examination in situ of a testis devoid of most germ cells and by Northern analysis of RNA from an adult Sertoli cell-enriched preparation. However, using either approach the amount of mRNA in Sertoli cells was considerably lower than that in pachytene spermatocytes. Cytochrome C oxidase has thirteen subunits, three of which (I-III) are encoded on the mitochondrial genome. The physiological significance of the high levels of expression of COX II mRNA in pachytene spermatocytes and its stage-dependent changes is unknown but they presumably reflect requirements for alterations in energy demand as these cells enter the final stages of meiosis. The stage-dependent differences in expression of COX II mRNA in pachytene spermatocytes may also explain differences in the susceptibility of these cells to depletion by MAA.

CDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis

A cDNA library of RNA from barley leaves inoculated with Erysiphe graminis was screened using labelled cDNA enriched for specific sequences by subtractive hybridization against RNA from non-inoculated leaves. This resulted in isolation of several clones representing pathogen induced genes. By cross-hybridization and sequence analysis, one of the cDNAs (pBT6-3) was found to be a partial clone representing a putative peroxidase, for which a full-length cDNA clone (pBH6-301) was subsequently isolated. The predicted amino acid sequence revealed a 21 amino acid signal peptide and a 294 amino acid mature protein (31 kDa) and shows 56 % amino acid identity to a basic peroxidase from turnip, 89 % to a putative peroxidase from wheat, but only 38% to the amino acid sequence derived from the cDNA clone (pcD1311) of a second putative barley peroxidase expressed in leaves. Northern blot analysis showed that the pBT6-3 (pBH6-301) transcript is elevated as early as 4 h after inoculation with E. graminis f. sp. hordei and that two maxima in transcript levels appear, which can be correlated with penetration attempts by the fungus. The amount of the pcD1311 transcript was also found to increase in inoculated leaves but at a later time point.

Tissue-specific expression of an 11 beta-hydroxysteroid dehydrogenase with a truncated N-terminal domain. A potential mechanism for differential intracellular localization within mineralocorticoid target cells.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) is thought to confer specificity on the nonselective Type I adrenocorticoid receptor by converting glucocorticoids to receptor-inactive metabolites in mineralocorticoid target tissues. S1 nuclease analyses using a rat liver 11-HSD probe demonstrated tissue-specific expression of the 5' region of the 11-HSD gene in the liver, lung, and kidney not evident in previous studies. Renal tissue contained a unique protected species which mapped to a position within the coding region, consistent with a divergence in liver and kidney protein sequences. Screening of a rat kidney cDNA library resulted in the isolation of several clones (11-HSD1B) noncolinear in their 5' regions with the liver sequence (11-HSD1A). Nucleic acid sequence analysis showed that the divergent clones code for a protein lacking a 26-amino acid NH2-terminal putative membrane-spanning signal peptide. The deletion of the leader sequence from the microsomal 11-HSD1A protein may result in a nuclear localization of the 11-HSD1B isoform. The renal 11-HSD1A and 11-HSD1B species increased coordinately during ontogeny and in parallel with the developmental surge in glucocorticoids. At least three alternate sites of polyadenylation were found to be utilized by the 11-HSD gene. Southern blot analysis showed the presence of a single gene in the rat. This study shows the expression of a kidney-specific 11-HSD isoform which may protect the Type I adrenocorticoid receptor from occupation by glucocorticoids in the nucleus of a mineralocorticoid target cell.

CDNA sequence of Xenopus laevis bone morphogenetic protein 2 (BMP-2)

Screening of a Xenopus laevis ovary cDNA library with an oligonucleotide derived from the activin A sequence led to the isolation of several clones belonging to the TGF-beta supergene family. One of these clones codes for the Xenopus bone morphogenetic protein 2 (BMP-2). The deduced protein contains 398 amino acids with a predicted Mr of 45,581. Within the C-terminal 114 amino acids constituting the mature, biologically active protein there are only four exchanges (three of them are conservative ones) as compared to the corresponding human sequence.

Schistosoma mansoni: Cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a λgt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed β-galactosidase/ S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than β-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant λ clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.

NK sensitivity, H-2 expression and metastatic potential: analysis of H-2Dk gene transfected fibrosarcoma cells.

We have used the 3-Methylcholanthrene induced T-10 fibrosarcoma tumour cell system (H-2b xH-2k)F1 to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non-metastatic and NK sensitive IC9 cells (Db+, Dk-, Kb-, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumour cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Dk+, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Retransfection of 'Dk' 'loss' variants with the H-2Dk gene, resulted in the isolation of several clones expressing a wide range of metastatic phenotypes but maintained sensitivity to NK. These results indicate that the H-2D region of the MHC and or closely linked genes may be involved in the complex interrelationship between target susceptibility to NK and metastasis.

Isolation of a multigene family containing human α-tubulin sequences

Screening of two human genomic libraries with a chicken α-tubulin complementary DNA probe has resulted in the isolation of several clones containing a common hybridizing region. Restriction mapping of the cloned fragments reveals that, while the majority of sites are retained in each clone, there are a number of differences, including several within the gene-containing region. These differences cannot be ascribed solely to restriction-site polymorphism, and therefore reflect the existence of a family of closely related α-tubulin genes.