The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins.

The two beta-tubulin genes of the unicellular green alga Chlamydomonas reinhardtii are expressed coordinately after deflagellation and produce two transcripts of 2.1 and 2.0 kilobases. Full-length cDNA clones corresponding to the transcript of each gene were isolated. DNA sequences were obtained from the cDNA clones and from cloned tubulin gene fragments. Both genes contained 1,332 base pairs of coding sequence, with only 19 nucleotide differences between the genes. Because all the differences occurred at the third base position of a codon and did not change the predicted amino acid sequence, we concluded that both beta-tubulin genes code for the same protein of 443 amino acids. The predicted amino acid sequence is 89 and 72% homologous with beta-tubulins from chicken and yeast cells, respectively. Each gene had three intervening sequences, which occurred at identical positions. Although the first two intervening sequences were not conserved between the two genes, the nucleotide sequence of the third intervening sequence was 89% conserved between the genes. The codon usage in the tubulin genes of C. reinhardtii was very biased: only 37 different codons were used. Striking differences occurred between the codons used in these nuclear genes and C. reinhardtii chloroplast genes.

The genomic structure and promoter analysis of the human ABF-1 gene.

The human ABF-1 gene is expressed in activated B-cells and Epstein-Barr virus-immortalized lymphoblastoid cell lines. ABF-1 represents the only member belonging to the basic helix-loop-helix (bHLH) family of transcription factors whose expression pattern is restricted to B-cells. ABF-1 forms heterodimeric complexes with E2A to modulate gene transcription. We report the cloning and characterization of the human ABF-1 gene and the promoter region. The gene spans more than 3 kb and contains two exons. Exon 1 contains 274 bp of a 5'-untranslated sequence (UTR) while exon 2 contains 1097 bp of 3'-UTR. Promoter analysis of the 5'-flanking region revealed no apparent B-cell-restricted control elements within approximately 700 bp, but clearly demonstrated the presence of a functional minimal promoter residing immediately upstream of the transcription start site. Analysis of the region containing the minimal promoter activity identified no CCAAT or TATA sequence. Lastly, we have assigned the ABF-1 gene to human chromosome 8q21.1 using fluorescence in situ hybridization (FISH). The cloning of the human ABF-1 gene will facilitate further biochemical and genetic studies of its function in the regulation of B-cell differentiation.

Expression of a specific protein in spontaneously metastatic fibrosarcoma cell lines and its enhanced synthesis by growth on laminin or fibronectin.

A panel of tumor cell lines and clones was generated by selecting for different metastatic capacity in 3AM fibrosarcoma cells. These cell lines were exposed to substrata of various purified extracellular matrix (ECM) proteins and labeled in culture with [35S]methionine. Following analysis by two-dimensional polyacrylamide gel electrophoresis the profiles of total cellular proteins produced by the cell lines displaying different phenotypes were examined. The presence of a specific protein, p54, was related to the cellular metastatic potential, and the synthesis of p54 was influenced by growth on extracellular matrix components. The amount of p54 was minimal and non-inducible in tumor cell lines exhibiting two different phenotypes: (1) experimentally metastatic (EM) and (2) transformed, non-tumorigenic (NTT) cell types. In contrast, all of the five different cell lines capable of both spontaneous and experimental metastasis (SEM), produced p54 either constitutively or through induction by growth on ECM protein substrata of either laminin of fibronectin, but not collagen type IV. These data suggest that the p54 protein may be a unique biochemical marker associated with spontaneous metastatic cell types.

Chlamydomonas reinhardtii tubulin gene structure.

DNA sequencing studies have provided a picture of the total information available at the gene level for tubulin production in C. reinhardtii. The data indicates that diversity at the gene level is very limited and that all the microtubules in the cell are composed of a very similar set of tubulins. These studies contrast with similar studies of S. pombe alpha-tubulin genes and chicken beta-tubulin genes that show much heterogeneity among members of the same gene family. Further studies will be needed to investigate whether the high degree of conservation of tubulin genes is unique or common among lower eukaryotes, and what mechanisms are used to maintain homogeneity in C. reinhardtii tubulin gene families. Our DNA sequence analysis, in addition to the work of Brunke et al., has provided information on the noncoding, and possibly regulatory, portions of the tubulin genes. For example, the promoter regions of the 4 tubulin genes share a consensus sequence of 16 nucleotides upstream of the TATA box. This sequence could be involved in regulating the coordinate expression of the genes. Although little homology exists generally in the noncoding region of the genes, striking homology between the third IVS in each beta-tubulin gene is observed. Small elements homologous to the beta-tubulin IVS 3 also exist in the second IVS of each alpha-tubulin gene. In addition, considerable homology in the 5' noncoding portion of the alpha-tubulin transcripts has been noted. These homologies may be the result of recent gene conversion events, and may not have functional significance. The possibility, however, must also be considered in future experiments that these elements may play a role in regulating the expression of the tubulin genes.

Differential evolution in chromosomal composition during multiple cycles of subcutaneous and intravenous metastasis.

It has been proven that multiple cycles of metastasis can improve the metastatic potential and homing specificity of a tumor cell population. In the present study, verification of genetic alterations during changes in metastatic behavior was done by analyzing the chromosome composition of a methylcholanthrene induced murine fibrosarcoma, 3AM during multiple cycles of subcutaneous (SC) and intravenous (IV) metastasis. After 10 cycles of SC metastasis, a cell type, 7B, with a small t(19;19)(A;A) metacentric marker chromosome was enriched from 4% in the original population to 90% in FIOR. However, when the tumor cells were injected IV rather than SC, no enrichment of the 7B cell type was observed. Instead, a cell type AX with a large t(14;19)(E5;A) acrocentric marker chromosome was enriched from 1% in the parental population to 76% in F1OIV after 10 cycles of IV metastasis. The polyploid dominant FIOIV was found to be extremely high in IV metastasis (411 foci/lung) but low in SC metastasis (48 foci/lung). The diploid dominant FIOR appears to be high in both SC (163 foci/lung) and IV (301 foci/lung) metastasis. The data obtained suggest that metastasis will lead to the selection of specific preexisting cell types, and the type of cell selected will depend on the route of metastasis. Furthermore, during metastasis, new cell types may also be produced de novo through chromosomal structural and numerical aberrations.

Inhibition of the adaptive response of human lymphocytes to very low doses of ionizing radiation by the protein synthesis inhibitor cycloheximide.

When human lymphocytes are preirradiated with 1 cGy of X-rays, the cells become less sensitive to subsequent exposures to high doses of about 150 cGy in that approximately one-half as many chromatid aberrations are induced as expected. This adaptation has been attributed to the induction of repair enzymes (proteins) some 4-6 h after the initial low-dose exposure. Experiments have now been carried out showing that application of the protein synthesis inhibitor cycloheximide at this time, but not earlier, prevents the adaptive response.

Cell electroporation is a highly efficient method for introducing restriction endonucleases into cells.

Restriction endonucleases that make either blunt- or cohesive-end DNA double-strand breaks can induce chromosome aberrations. We have used cell electroporation with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes. The introduction of restriction enzymes by this method resulted in extremely high frequencies (greater than 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation. Cell electroporation by itself caused no increase in aberrant chromosomes and had only a slight effect on cell survival.

Structural organization, DNA sequence, and expression of the calmodulin gene.

Calmodulin is encoded in Chlamydomonas reinhardtii by a single gene that 1) has multiple intervening sequences, 2) has 5' structural motifs that are phylogenetically conserved, 3) contains 5' sequences that are similar to those found in genes of some transforming, cytoskeletal, and stress-response proteins, and 4) produces at both life cycle stages, a single size class of mRNA and proteins that are identical in amino acid sequence. Based on the amino acid sequence of calmodulin from the vegetative phase of the life cycle, synthetic oligonucleotide probes, containing inosine in order to reduce codon redundancy, were used to detect and isolate cloned cDNAs coding for the gametic phase calmodulin. The complete DNA sequence was elucidated and shown to code for a protein identical to the vegetative phase protein. Analysis of the production of calmodulin mRNA indicates that protein production is under quantitative regulation and possibly coupled with the synthesis of other proteins in the flagellar apparatus. The full length cDNA was used to isolate overlapping genomic clones that include the entire calmodulin transcriptional unit and 5' regulatory sequences. The complete DNA sequence of the gene, including all intron sequences, was elucidated. The DNA sequence of the coding regions shows some phylogenetic conservation. Finally, there are regions of 5' sequence reminiscent of sequence motifs recently identified as binding sites of transcriptional regulatory proteins. Overall, these studies suggest possible molecular genetic relationships between calmodulin, a transducer of intracellular calcium signals, and other proteins involved in eukaryotic cell structure, motility, and homeostasis.

The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains.

We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DC1v, maps at the amino-terminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMV-infected cells by flow cytometry with antibodies to the amino- and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.

Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.

Cleavage in embryos of the sea urchin Arbacia punctulata consists of eight very rapid divisions that require continual protein synthesis to sustain them. This synthesis is programmed by stored maternal mRNAs, which code for three or four particularly abundant proteins whose synthesis is barely if at all detectable in the unfertilized egg. One of these proteins is destroyed every time the cells divide. Eggs of the sea urchin Lytechinus pictus and oocytes of the surf clam Spisula solidissima also contain proteins that only start to be made after fertilization and are destroyed at certain points in the cell division cycle. We propose to call these proteins the cyclins.

Fluorescence in situ hybridization mapping of human chromosome 19: cytogenetic band location of 540 cosmids and 70 genes or DNA markers.

We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19 cosmids on other chromosomes is also reported. Colony hybridization identified 142 cosmids that contain sequences representing genes or DNA markers that map to chromosome 19. FISH mapping of these cosmids sublocalizes a total of 70 genes and DNA markers on chromosome 19, revises the previously published map assignments of 2 genes, and narrows the location of over 20 markers.

Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes.

We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.