Degradation of nucleosome-associated centromeric histone H3-like protein CENP-A induced by herpes simplex virus type 1 protein ICP0.

Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. Here, we demonstrate that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments we show that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation.

Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.

BACKGROUND: The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells. RESULTS: The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells. CONCLUSIONS: The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.

Isolation and comparison of natural and recombinant human CENP-A autoantigen.

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of the bona fide antigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.

Persistent pulmonary hypertension of the newborn and smoking and aspirin and nonsteroidal antiinflammatory drug consumption during pregnancy.

OBJECTIVE: Prenatal causation of persistent pulmonary hypertension of the newborn (PPHB) is suggested by a specific pattern of pulmonary vascular remodeling observed immediately after birth in some infants with fatal PPHN. The goal of this study was to determine whether PPHN is associated with fetal exposure to: (1) tobacco and marijuana smoking (ie, contributors to fetal hypoxemia), (2) consumption of aspirin and other nonsteroidal antiinflammatory drugs (ie, inhibitors of prostaglandin synthesis), and (3) cocaine use (ie, a contributor to vasospasm). DESIGN: Case-control interview study. SETTING: Two Harvard-affiliated newborn intensive care units. PARTICIPANTS: Mothers of case infants who had PPHN or who met criteria for the referent group. INTERVENTIONS: During July 1985 through April 1989, we interviewed mothers of 103 infants with PPHN and 298 control infants. Because of potential selection bias that might result from recruiting only inborn control infants even though two-thirds of cases were outborn, separate analyses compared the 103 total and 35 inborn infants with PPHN with the 298 inborn control infants. Multivariate analyses were used to adjust for potential confounding factors, including maternal education and Medicaid health insurance (ie, two markers of socioeconomic status), other antenatal factors found to be associated with PPHN (ie, maternal urinary tract infection and diabetes mellitus), and the infant's sex. MAIN OUTCOME MEASURES: Self-reported use or consumption of tobacco, marijuana, cocaine, aspirin, and other nonsteroidal antiinflammatory drugs during pregnancy. RESULTS: The adjusted odds ratios (and 95% confidence intervals) for maternal pregnancy exposures to the factors of principal interest among the total study population were: aspirin, 4.9 (1.6-15.3); and nonsteroidal antiinflammatory drugs, 6.2 (1.8-21.8); for the inborn group they were aspirin, 9.6 (2.4-39.0); and nonsteroidal antiinflammatory drugs, 17.5 (4.3-71.6). Although the association between tobacco smoking during pregnancy and PPHN was elevated in univariate analyses, with odds ratios (and 95% confidence intervals) of 2.0 (1.2-3.4) and 1.3 (0.6-3.3) for total and inborn populations, respectively, the relationship was not significant after adjustment for all other factors in the final logistic regression model. Acknowledged illicit drug use was too infrequent (3.2%) to evaluate. CONCLUSION: Maternal consumption of nonsteroidal antiinflammatory drugs and aspirin during pregnancy or the reasons these drugs were ingested seem to contribute to an increased risk of PPHN.

Detection of anticentromere antibodies using recombinant human CENP-A protein.

OBJECTIVE: To evaluate CENP-A reactivity with anticentromere antibodies (ACA) using recombinant protein (rCENP-A). METHODS: Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the ACA+ sera studied (n = 38), 95% were positive when tested against the rCENP-A in the ELISA system. Of the ACA- sera (n = 100), only 2% gave false-positive results in the assay. There was good correlation between the recombinant and bona fide antigens in assaying for ACA reactivity. CONCLUSION: CENP-A is a significant ACA target. The availability of the rCENP-A assay is a valuable adjunct to the previously described rCENP-B assay in analyses of the clinical significance of ACA.

Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere.

Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.

LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested. An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies. Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1. 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3. Affinity purified LKM-1 antibodies did not recognize C100-3. However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein. Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies.

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins.

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5' and 3' flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.

Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products.

A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well-characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as might occur with antigens that are components of subcellular particles.

Patients with type II autoimmune hepatitis express functionally intact cytochrome P-450 db1 that is inhibited by LKM-1 autoantibodies in vitro but not in vivo.

Liver-kidney microsomal-1 autoantibodies characterize a subgroup of autoimmune chronic active hepatitis. The liver antigen of liver-kidney microsomal-1 antibodies has been identified as cytochrome P450 db1, a microsomal enzyme catalyzing the oxidative metabolism of more than 20 drugs, including debrisoquine, sparteine and bufuralol. A genetic polymorphism (debrisoquin-sparteine polymorphism) is responsible for the lack of P450 db1 protein in the livers of 5% to 10% of Caucasians, leading to impaired drug metabolism and a distinct poor metabolizer phenotype. We investigated whether liver-kidney microsomal-1 positive autoimmune chronic active hepatitis patients express functionally intact P450 db1 in their livers. In four patients with liver-kidney microsomal-1 positive chronic active hepatitis, but not in five patients with various liver-kidney microsomal-1 negative liver diseases, the presence of circulating liver-kidney microsomal-1 antibodies was confirmed by immunofluorescence, radioimmunoassay and immunoblotting analysis using recombinant P450 db1. Moreover, only sera from liver-kidney microsomal-1 positive autoimmune chronic active hepatitis patients strongly inhibited the enzymatic activity of P450 db1 in human liver microsomes in vitro. Immunoblotting detected 50-kd P450 db1 protein in liver biopsy specimens from all patients. The in vivo function of P450 db1 was investigated by determining the metabolic ratio for sparteine and its 2-dehydro and 5-dehydro metabolites in 12-hr urine samples after oral administration of sparteine sulfate. In vivo P450 db1-mediated drug metabolism was of the extensive metabolizer phenotype and did not differ significantly between liver-kidney microsomal-1 positive (metabolic ratio = 1.15 +/- 0.32) and liver-kidney microsomal-1 negative (metabolic ratio = 1.18 +/- 0.48) patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a cDNA clone encoding the 60-kD component of the human SS-A/Ro ribonucleoprotein autoantigen.

SS-A/Ro is a nucleocytoplasmic ribonucleoprotein (RNP) particle that is a common target of autoimmune response in Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Previously, SS-A/Ro has been shown to be composed of at least two polypeptide antigens of 60 and 52 kD noncovalently associated with a set of small RNAs, designated Y1-Y5. A serum from an SS patient was selected to screen a lambda gt11 cDNA library constructed from human T cell lymphoblastic leukemia (MOLT-4) mRNA. An immunoreactive clone was isolated that possessed a 1.8-kb cDNA insert. In vitro transcription and translation of the cDNA resulted in the synthesis of a 57.5-kD polypeptide which was specifically immunoprecipitated by SS-A/Ro antisera. The identity of the cDNA encoded protein as the 60-kD SS-A/Ro antigen was established by proteolytic peptide mapping of the cDNA-encoded protein and the 60-kD HeLa cell antigen. The sequence of the cDNA shows that the 60-kD SS-A/Ro protein possesses both RNA binding protein consensus sequences and a single zinc-finger motif. Recombinant SS-A/Ro antigen produced in bacteria proved to be a sensitive and specific reagent for detection of anti-SS-A/Ro antibodies in patient sera. The availability of the 60-kD SS-A/Ro cDNA will enable detailed analysis of the molecular structure and function of the SS-A/Ro RNP particle and its role in autoimmune pathology.

Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1.

Type 1, liver kidney microsomal autoantibodies (LKM-1) are associated with a subgroup of idiopathic autoimmune type, chronic active hepatitis (CAH). The antigenic specificity of LKM-1 autoantibodies from 13 patients was investigated by immunoblot analysis of human liver microsomal proteins. Polypeptides of 50, 55, and 64 kD were detected with these antisera. A high titer LKM-1 serum was selected to screen a human liver lambda gt11 cDNA expression library, resulting in the isolation of several complementary (c)DNA clones. Autoantibodies affinity purified from proteins expressed by two of the immunopositive cDNA clones, HLD8.2 and HLD13.2, specifically react with a 50-kD protein of human liver microsomes and display immunofluorescence staining of the proximal renal tubular epithelia characteristic of LKM-1 sera. Determination of the sequence of HLD8.2 revealed that it encodes a recently described cytochrome P450db1. A bacterial fusion protein constructed from HLD8.2 proved to be a specific and sensitive diagnostic reagent. All sera from patients with LKM-1 positive liver disease react with this fusion protein. No reaction was seen, however, for sera from patients with other types of autoimmune liver diseases, viral hepatitis, systemic immunological disorders, or healthy controls.

Arterial blood gas derangements associated with death and intracranial hemorrhage in premature babies.

We evaluated to what extent acidosis and alkalosis and their respiratory and metabolic components during the first 12 hours of life occurred prior to early neonatal death and postnatal intracranial hemorrhage among 206 low birth weight, intubated premature babies participating in a clinical trial of phenobarbital prophylaxis for intracranial hemorrhage. Time-weighted indices included the time each baby spent with abnormal values of pH, PaCO2 and HCO3-. Babies whose birth weight was less than 1 kg suffered adversities associated with prolonged pH less than 7.35. Heavier birth weight babies were at increased risk of adversity if their pH fell below 7.2. Babies who were not severely acidotic initially, but became so within hours, were at prominently increased risk of death and hemorrhage. Babies who had a mild increase of PaCO2 between 45 and 60 mmHg were less likely to develop germinal matrix hemorrhage than their peers who had more severe hypercapnia. A time-weighted measure of metabolic deficit correlated with death, but not with hemorrhage. Prolonged exposure to pH greater than 7.55 was associated with reduced risk of subependymal/intraventricular hemorrhage and death, especially in babies below 1 kg birth weight. We conclude that acidosis is an antecedent of intracranial hemorrhage in low birth weight premature babies, that duration of exposure might convey important risk information, and that birth weight is a correlate of vulnerability to some pH disturbances.

Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen.

We have isolated a series of overlapping cDNA clones for approximately 95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is approximately 65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.

Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression.

We have determined the nucleotide sequence of the chicken beta 5 (c beta 5)-tubulin gene. The gene displayed the coding structure common to all previously studied vertebrate beta-tubulin genes and was divided into four exon sequences interrupted by three intervening sequences (located between codons 19 and 20, within codon 56, and within codon 93). Comparison of the predicted polypeptide sequence encoded by c beta 5 with those of four other available chicken beta-tubulin sequences revealed that c beta 5 encoded a highly divergent beta-tubulin polypeptide isotype which was distinguished from previously known sequences primarily by two discrete variable sequence domains. However, c beta 5 uniquely shared identity in 16 residue positions with another divergent chicken beta-tubulin gene, c beta 4. These common sequences distinguished c beta 4 and c beta 5 from the remaining three chicken beta-tubulin genes. Analysis of the expression of c beta 5 and c beta 4 revealed a strikingly complementary pattern of gene expression: c beta 5 was expressed in a wide variety of cell and tissue types but not in neurons, whereas c beta 4 expression was detected uniquely in neuronal cells. Overall, these findings suggest the existence of two divergent families of beta-tubulin sequences in the chicken and further raise the possibility that the complementary expression of the c beta 4 and c beta 5 genes may fulfill a requirement for the presence of a divergent beta-tubulin polypeptide isotype in all cell types.

Sequence and expression of the chicken beta 3 tubulin gene. A vertebrate testis beta-tubulin isotype.

We report the determination of the complete DNA sequence for c beta 3, a chicken beta-tubulin gene which we show to be the dominant beta-tubulin expressed in testis. Like all previously studied vertebrate beta-tubulin genes, the gene is divided into four exon sequences interrupted by three intervening sequences (located between amino acids 19 and 20, within codon 56, and within codon 93). Analysis of the program of expression of this gene indicates that it encodes the dominant chicken testis beta-tubulin, although it is also expressed at lower levels in a wide variety of cell and tissue types. Comparison of the predicted polypeptide sequence for c beta 3 with four other available chicken beta-tubulin genes confirms our earlier suggestion that within an otherwise conserved framework, sequences within two variable region domains serve to define specific beta-tubulin polypeptide isotypes. The data indicate that the c beta 3 gene encodes a unique beta-tubulin isotype which diverges from the dominant neuronal beta-tubulin isotype in 18 of 445 residues (4%). Although the protein coding regions of the c beta 3 gene are highly homologous to the chicken c beta 1, c beta 2, c beta 4, and c beta 5 genes previously reported by us, no significant sequence homology with these previously analyzed genes is discernible in the 5'- or 3'-untranslated region sequences, in the intervening sequences, or in the presumptive transcriptional promoter sequences.

Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes.

We report the determination of the complete sequences for two chicken beta tubulin genes, beta 3 and beta 5. Taken with the previously published efforts, we have determined the primary structures of five of the seven beta tubulin genes in this vertebrate species. A comparison of these sequences unambiguously reveals that amino acid sequence variations among different beta tubulin gene products are distinctly clustered within an otherwise highly conserved framework of the beta tubulin molecule. To determine the extent to which this pattern of structural heterogeneity is conserved among vertebrates, we have isolated novel beta tubulin sequences from human and mouse cDNA libraries and compared these and all other known vertebrate beta tubulin sequences with the family of chicken polypeptide sequences. What emerges from such comparison is the recognition of distinct, evolutionarily conserved isotypes of beta tubulin that are distinguished primarily by their characteristic carboxyl-terminal variable region sequence and, to a lesser extent, by sequence in an amino-terminal variable domain as well. These correlations represent a convincing demonstration that multiple beta tubulin genes in vertebrates encode a family of closely related but structurally distinct beta tubulin isotypes and further serve to define the sequences of four classes of polypeptide isotypes that constitute that family.

Apparent gene conversion between beta-tubulin genes yields multiple regulatory pathways for a single beta-tubulin polypeptide isotype.

We have determined the complete nucleotide sequences of two chicken beta-tubulin genes, beta 1 and beta 2. These genes display an unusual pattern of segmental homology which indicates that they originally arose by gene duplication and have subsequently coevolved by a process that included localized gene conversion or intergenic recombination. Since the beta-tubulin polypeptides encoded by the two genes are virtually identical (99.5%), particularly in the major beta-tubulin isotype defining regions, they almost certainly constitute a single isotypic class of beta tubulin. However, the regulatory properties of the two genes are highly divergent as indicated by analysis of their patterns of expression in different chicken cell types. beta 1 is the major transcript detected in skeletal muscle myoblasts, whereas beta 2 is the major beta-tubulin transcript in cultured sympathetic neurons. The existence of these two genes appears to derive from a regulatory requirement whereby the expression of a single tubulin isotype is mediated through different regulatory programs in development and differentiation. These results thus provide direct experimental support for the hypothesis that gene conversion and intergenic recombination play an important role in evolution by uncoupling the evolution of structural genes from the regulatory sequences which control them.

Sequence of a highly divergent beta tubulin gene reveals regional heterogeneity in the beta tubulin polypeptide.

The nucleotide sequence of a chicken genomic DNA segment containing the chicken beta 4 tubulin gene has been determined. The predicted amino acid sequence of beta 4 is surprisingly divergent from that of the chicken beta 2 gene that encodes the dominant neural beta tubulin. beta 4 differs from beta 2 at 36 residue positions and encodes a polypeptide that is four amino acids longer, yielding a divergence of 8.9% between the two beta tubulin isotypes. While many of the amino acid substitutions are conservative, several involve significant alteration in the physiochemical properties of the residue. Furthermore, the amino acid substitution positions are not randomly located within the primary sequence but are distinctly clustered: major divergence occurs in the carboxy-terminal region beyond residue 430 and within the second protein coding exon segments of the genes. In addition, large regions of absolute sequence conservation are also present. Certain sequences within the heterogeneous regions are conserved in other species, indicating that these regions are under positive evolutionary selection pressure and are therefore probably essential for some aspect of beta-tubulin function. These findings strongly suggest that regional amino acid sequence heterogeneity may play an important role in the establishment of functionally differentiated beta tubulin polypeptides.