Nucleotide sequence and expression of the human skeletal alpha-actin gene: evolution of functional regulatory domains.

Regulation of the actin multigene family involves the recognition of regulatory sequences that specify the tissue type and developmental program of expression for each actin isotype. In order to investigate the underlying regulatory mechanisms, the human skeletal alpha-actin gene and its 5' regulatory region have been cloned and sequenced. This actin gene has seven exons; there is one large intron in the 5' untranslated region which is characteristic of the actins and many muscle-specific genes. The 5' flanking sequences are sufficient to direct tissue-specific and differentiation-regulated expression when transfected into the heterologous rat L8 myogenic cells, indicating a highly conserved regulatory system. The DNA sequence was compared to that of other actin genes, and several regions of sequence similarity were identified, particularly within regions known to be important for gene expression. Most notable among the conserved sequences are the CC(A/T rich)6GG (CArG box) motifs which have demonstrated interactions with trans-acting transcriptional factors. This same motif has been identified in several other genes and in some also serves as a binding site for transcription regulatory factors.

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.

Cytoskeletal actin gene families of Xenopus borealis and Xenopus laevis.

We have sequenced the coding and leader regions, as well as part of the 3' untranslated region, of a Xenopus borealis type 1 cytoskeletal actin gene [defined according to the arrangement of acidic residues at the N-terminus; Vandekerckhove et al. (1981) J Mol Biol 152:413-426]. The encoded amino acid sequence is the same as the avian and mammalian beta (type 1) cytoskeletal actins, except for an isoleucine at position 10 (as found in the mammalian gamma cytoskeletal actins), and an extra amino acid, alanine, after the N-terminal methionine. Five introns were found, in the same positions as those of the rat and chicken beta-actin genes. The 5' and 3' untranslated regions resemble those of the human gamma (type 8) cytoskeletal actin gene more closely than the mammalian beta genes. Primer extension showed that this type 1 gene is transcribed in ovary and tadpole. Sequencing of primer extension products demonstrated two additional mRNA species in X. borealis, encoding type 7 and 8 isoforms. This contrasts with the closely related species Xenopus laevis, where type 4, 5, and 8 isoforms have been found. The type 7 isoform has not previously been found in any other species. The mRNAs of the X. borealis type 1 and 8 and X. laevis type 5 and 8 isoforms contain highly homologous leaders. The X. borealis type 7 mRNA has no leader homology with the other mRNA species and, unlike them, has no extra N-terminal alanine codon. The evolutionary implications of these data are discussed.

Nucleotide sequence of the human gamma cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes.

Two distinct, but iso-coding, gamma non-muscle actin cDNAs were isolated from an SV40-transformed human fibroblast library. The complete nucleotide sequence of the human gamma non-muscle actin cDNAs indicates that they may have arisen from polymorphic alleles. By using genomic DNA and cellular RNA transfer blots, we demonstrate that the 3' untranslated region (UTR) of the gamma actin mRNA consists of an evolutionarily conserved 5' and more divergent 3' segments. In fact, the conserved segment of the 3' UTR detects a single-copy sequence in the chicken genome and a 20S RNA transcript in chicken non-muscle tissues. The coding regions of these cDNAs were compared with those of other vertebrate non-muscle actin genes. Surprisingly, the percentage of silent base substitutions between the human beta and gamma actin coding regions is anomalously low and indicates greater sequence conservation than would be expected for a gene pair which arose during pre-avian evolution. We discuss gene conversion and recent selective pressure as possible explanations of the apparently anomalous evolution of the gamma non-muscle actin gene.

Histone gene clusters of the newt notophthalmus are separated by long tracts of satellite DNA.

The genomic organization of the histone genes of the newt Notophthalmus viridescens is described. Genes for the five proteins are clustered on a 9.0 kb segment of cloned DNA which is part of a homogeneous family of sequences containing 600--800 members per haploid genome. The 9.0 kb histone gene clusters are not adjacent in the genome, but are separated from neighboring clusters by up to 50 kb or more of cluster spacer sequences; some or all of these spacer sequences are members of a predominantly centromeric satellite DNA with a 2235 bp repeating unit.

Characterization of a cloned histone gene cluster of the newt Notophthalamus viridescens.

We report the cloning and characterization of a histone gene cluster of the newt Notophthalamus viridescens. Fragments containing newt histone genes were identified in whole genome Southern blots; these fragments were cloned into a bacteriophage lambda cloning vector constructed for this purpose. The positions of most of the histone genes were determined by hybridizing subcloned sea urchin histone genes to digests of the cloned newt gene cluster. The position of each gene was verified, and its polarity determined by sequencing a portion of each. The order of the genes in the cloned segment is H1-H3-H2B-H2A-H4, with each of the genes but H2B being transcribed in the same direction. Subcloned segments of the histone gene repeat were used to determine the size of each newt oocyte histone mRNA.

Histone genes are located at the sphere loci of newt lampbrush chromosomes.

In situ hybridization experiments on mitotic and lampbrush chromosomes show that histone genes and an associated 222 bp repeated sequence, satellite 1, are located at or near the sphere loci of chromosomes 2 and 6 of the newt Notophthalmus viridescens. During the lampbrush chromosome stage, transcripts of the histone genes and of satellite 1 occur on loops associated with the spheres. Histones genes are located at sites on the mitotic chromosomes of Triturus cristatus and T. alpestris which are consistent with the known positions of spheres on their lampbrush chromosomes.

Satellite DNA is transcribed on lampbrush chromosomes.

During the lampbrush stage of oogenesis there is widespread transcription, and it has been estimated that the total amount of DNA transcribed may be an order of magnitude greater than that required to produce the necessary functional RNA for the oocyte. We therefore considered it likely that some of the transcribed sequences have little, if any, translational significance, and may include both middle repetitive and highly repeated, or satellite, sequences. Satellite DNA is generally defined as rapidly reannealing DNA which has a short basic sequence that is repeated millions of times in the genome, usually in tandem arrays. The short repeated length, coupled with the organisation of satellite sequences in high order molecular weight tandem arrays in heterochromatic regions, have been put forward as reasons for supposing that this type of DNA is not normally transcribed. We report here that we have looked for and found evidence of transcription of satellite DNA on lampbrush loops in oocytes of the crested newt, Triturus cristatus carnifex.

Cytological evidence of transcription of highly repeated DNA sequences during the lampbrush stage in Triturus cristatus carnifex.

Highly repeated, or satellite, DNA fractions have been isolated from total Triturus cristatus carnifex DNA by renaturation kinetics, caesium salt centrifugation and restriction endonuclease digestion. We have shown by DNA/DNA in situ hybridisation and autoradiography that all of these probes bind to C-band positive regions on mitotic or lampbrush chromosomes of T.c. carnifex. Under conditions of DNA to RNA-transcript in situ hybridisation labelled satellite DNA binds to nascent RNA transcripts that are still associated with the DNA axes of many lampbrush loops. The majority of the loops that label heavily in these experiments are located on the long arms of chromosome I, a region previously shown to be rich in highly repeated DNA and to have many of the properties of heterochromatin. These satellite DNA probes also label many loops on a comparable chromosome region in T. marmoratus, a species closely related to T. cristatus. However, in DNA/RNA-transcript hybrids to other more distantly related species of Triturus, there are no chromosome regions that have the same concentration of labelled loop pairs as the long arms of T.c. carnifex and T. marmoratus, although some loop pairs do label. We have cloned two satellite sequences in pBR322, and have obtained the same results using these pure probes as we obtained using satellite probes isolated by other techniques. These results demonstrate unequivocally that satellite DNA is transcribed on lampbrush chromosomes during oogenesis in crested newts.