Cloned ribosomal RNA genes from chloroplasts of Euglena gracilis.

Fragments of Euglena chloroplast DNA generated by endonuclease R-Eco RI were separated by agarose-gel electrophoresis into 24 distinct bands. At least five fragments contain sequences complementary to chloroplast ribosomal RNA, Most of the Eco RI fragments have been cloned in a plasmid of Escherichia coli. Three of the cloned fragments were shown to contain chloroplast ribosomal RNA sequences by DNA-RNA hybridization.

Tissue-specific genes for respiratory proteins.

All but one of the mitochondrial respiratory complexes are composed of products of both the mitochondrial and the nuclear genomes. The recent isolation of cDNAs for several nuclear-encoded respiratory proteins reveals that some of them are present in at least two forms. Although some of these forms are traditional in differing somewhat in amino acid sequence, a new class, termed silent isoforms, differs in the presequence but contains identical processed proteins. What are the roles of tissue isoforms in oxidative metabolism?

Beta 4 integrin transfection of UM-UC-2 (human bladder carcinoma) cells: stable expression of a spontaneous cytoplasmic truncation mutant with rapid loss of clones expressing intact beta 4.

The alpha 6 beta 4 integrin is a component of the hemidesmosome, the anchoring structure in the basal membrane of epithelial cells. alpha 6 beta 4 expression is frequently altered in neoplastic cells. It is sometimes lost and sometimes overexpressed, which suggests that disruption of normal function is involved in neoplastic transformation. To examine the effect of this integrin on the growth and behavior of malignant cells that have lost beta 4, we transfected a full-length beta 4 cDNA into the UM-UC-2 cell line that expresses alpha 6 but not beta 4. Although large numbers of clones were obtained when a control vector was used in the transfection, only 12 clones could be isolated that expressed beta 4. Of these, only two beta 4-positive clones, clones 8 and 11, persisted long enough for further study. Clone 8 cells initially expressed beta 4, but within 2 weeks, all positive cells were lost from the culture. Clone 11 persisted in culture and retained strong surface expression of alpha 6 beta 4. Biochemical analysis and Western blotting revealed that this clone contained a truncated form of beta 4 that had lost the distal cytoplasmic domain. We conclude that expression of wild-type beta 4 in UM-UC-2 inhibits cell growth, presumably by an integrin-mediated signaling pathway. Clone 11 escaped from normal signaling because the cytoplasmic domain, a region essential for basal polar localization, was lost. The alpha 6 beta 4 integrin appears to have tumor suppressor activity in epithelial tumors.

Sequence of the cDNA for the liver/non-muscle isoform of mouse cytochrome-c oxidase subunit VIII.

We have isolated and sequenced the cDNA for the liver (L) or non-muscle isoform of mouse cytochrome-c oxidase subunit VIII (COX VIII-L). Comparison of deduced COX VIII-L protein sequences from three mammalian species indicated that the human gene has sustained more amino acid replacement substitutions than either the mouse or the cow. The most highly conserved regions of this subunit are the N-terminal presequence and the C-terminal domain of the mature protein.

Structural organization of the bovine gene for the heart/muscle isoform of cytochrome c oxidase subunit VIa.

The bovine gene for the nuclear-encoded heart/muscle isoform of cytochrome c oxidase subunit VIa (COX6A1) was isolated from a library of bovine genomic DNA in lambda EMBL3 and sequenced. The gene spans 760 bp and comprises three exons and two small introns. Exon 1 encodes a 193 bp 5' untranslated region, a 12 amino acid presequence, and the first 12 amino acids of the mature COX VIa protein. Exon 2 encodes amino acids 13 to 58, and exon 3 amino acids 59 to 85 plus the 35 bp 3' untranslated region. Exons 2 and 3 are separated by a small intron of only 96 bp. All exon-intron boundaries matched the consensus splice junction sequences. COX6A1 transcripts are present in RNA from bovine heart but not brain. Primer extension and ribonuclease protection assays were used to map the 5' ends of COX6A1 transcripts in heart; both methods identified several clusters of transcription initiation sites, indicating that COX6A1 mRNA is heterogeneous at the 5' end. The proximal 5' flanking region is AT-rich and contains potential basal promoter elements, such as TATA and CCAAT boxes, associated with tissue-specific genes. A single consensus binding site for the muscle-specific transcription factor, MyoD1, was also located within this AT-rich region. The distal promoter region contained a perfect AP4 site plus potential binding sites for enhancer elements (NRF-1, Mt1, Mt3, and Mt4) proposed to regulate expression of genes for mitochondrial proteins.

The chicken cDNA for ornithine decarboxylase antizyme.

Two full length avian cDNAs for ornithine decarboxylase antizyme were isolated from a chicken cochlear cDNA library and differed in length through use of alternative poly(A) addition signals. The chick antizyme protein sequence predicted by translational frameshifting of the mRNA is 216 amino acids long and is more similar to Xenopus antizyme than to the mammalian protein.

Sequence of the cDNA for the heart/muscle isoform of mouse cytochrome c oxidase subunit VIII.

We have isolated and sequenced cDNAs for the heart/muscle (H) isoform of mouse cytochrome c oxidase subunit VIII (COX VIII-H). The deduced protein sequence enables us to compare the heart/muscle COX VIII isoforms from several species and to determine that the most highly conserved region of this subunit is the C-terminal domain.

Complete cDNAs for CDC42 from chicken cochlea and mouse liver.

CDC42 is a member of the ras superfamily of small GTP-binding proteins that are related through the highly conserved GTP-binding domain and are involved in signal transduction pathways. Two full-length CDC42 cDNAs have been isolated: a 2148-bp chick cochlea cDNA and a 2063-bp mouse liver cDNA. Each encodes a CDC42 protein of 191 amino acids. The avian CDC42 protein differs from the mouse at only one amino acid residue, a Thr for a Ser at position 185. Both CDC42 proteins are more similar to the ubiquitous human isoform originally isolated from placenta than to the isoform isolated from fetal brain. Using a probe from the 3' UTR of the mouse liver CDC42 cDNA, we demonstrated that the mouse gene is expressed in all tissues examined. Southern blot analysis of a mouse inter-specific backcross with this gene-specific probe identified at least three CDC42-like (Cdc42l) genes in the mouse genome. Cdc42l1 was mapped to distal mouse Chromosome 4, near Cappb1. Cdc42l2 mapped more proximal on Chromosome 4, whereas Cdc42l3 mapped to the X Chromosome.

Novel beta 3 isoform of the Na,K-ATPase beta subunit from mouse retina.

We isolated a full-length cDNA encoding a novel 278 amino acid beta subunit of Na,K-ATPase from a mouse retinal cDNA library. The highest sequence identity was to known beta 3 isoforms, identifying the protein as the mouse beta 3 subunit of Na,K-ATPase. Two transcripts, 1.75 kb and 2.1 kb, probably arise from use of alternative poly(A) addition signals.

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.

Human COX6A1 gene: promoter analysis, cDNA isolation and expression in the monkey brain.

The human COX6A1 gene encodes the ubiquitous isoform of cytochrome c oxidase (COX) subunit VIa (VIa-L), and is located in a CpG island on chromosome 12q24.2. We compared the COX6A1 gene with the published cDNA and several ESTs and concluded that subunit COX VIa-L is synthesized as a preprotein, as are other COX subunits. The same transcription start sites were identified by primer extension analysis of human brain and lymphoblastoid RNA. Analysis of the COX6A1 promoter revealed several conserved sequence elements found in other COX genes, namely binding sites for nuclear respiratory factor 1 (NRF-1), nuclear respiratory factor 2/GA binding protein (NRF-2/GABP), and ying-yang protein 1 (YY1). These conserved elements were shown to bind nuclear proteins from HeLa nuclear extracts. COX6A1 cDNA was isolated from a human brain cDNA library, and the sequence was identical to that of human liver. The expression of this gene was demonstrated by in-situ hybridization in monkey brain sections with our human brain cDNA. Monocular impulse blockade in adult monkeys induced a downregulation of COX6A1 expression in deprived visual neurons, suggesting that this subunit gene is regulated by neuronal activity.

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene.

We have isolated and analyzed 17 clones from a bovine genomic library in phage lambda Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.

A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene.

Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.

Novel use of a chimpanzee pseudogene for chromosomal mapping of human cytochrome c oxidase subunit IV.

We have isolated a chimpanzee processed pseudogene for subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1) by screening a chimpanzee genomic library in lambda Charon 32 with a bovine liver cDNA encoding COX subunit IV (COX IV), and localized it to a 1.9-kb HindIII fragment. Southern-blot analysis of genomic DNA from five primates showed that DNAs from human, gorilla, and chimpanzee each contained the 1.9-kb pseudogene fragment, whereas orangutan and pigtail macaque monkey DNA did not. This result clearly indicates that the pseudogene arose before the divergence of the chimpanzee and gorilla from the primate lineage. By screening Chinese hamster x human hybrid panels with the human COX4 cDNA, we have mapped COX4 genes to two human chromosomes, 14 and 16. The 1.9-kb HindIII fragment containing the pseudogene, COX4P1, can be assigned to chromosome 14, and by means of rearranged chromosomes in somatic cell hybrids, to 14q21-qter. Similarly, the functional gene, COX4, has been mapped to 16q22-qter.

Characterization and expression of a cDNA specifying subunit VIIc of bovine cytochrome c oxidase.

We have isolated a cDNA that encodes subunit VIIc of bovine cytochrome c oxidase (COX VIIc). The 325-bp cDNA contains sequences encoding the mature 47-amino acid (aa) polypeptide and a 16-aa presequence. The deduced aa sequence of the processed polypeptide is identical to that of the heart protein determined by aa sequencing. Northern-blot analysis reveals a single 525-nucleotide (nt) transcript in all tissues examined, whose levels vary with the corresponding respiratory activities in different tissues; thus, no evidence for isoforms of COX VIIc is seen in adult tissues. Southern-blot analysis of bovine genomic DNA digested with three different restriction enzymes reveals several bands that hybridize with the cDNA. We present here the sequence of one genomic region that contains a processed gene encoding COX VIIc. The genomic and cDNA nt sequences are 99% identical throughout the 189-bp open reading frame; the deduced aa sequences are identical. The sequence of the genomic clone suggests that the cDNA terminates prematurely at an EcoRI site in the 3'-untranslated region. We have compared COX VIIc cDNAs from cow, human and mouse, and find the presequence similarity among them to be 100% at the aa level.

Isolation of a cDNA clone encoding subunit IV of human cytochrome c oxidase.

We have isolated a full-length human liver cDNA clone specifying the nuclear-encoded subunit IV of the human mitochondrial respiratory chain enzyme, cytochrome c oxidase (COX; EC 1.9.3.1). The human cDNA clone is highly homologous to its bovine counterpart in the coding regions for both the mature polypeptide and the presequence, and the gene is evolving more slowly than that of any of the three mitochondrially encoded COX subunit genes. We find no preliminary evidence for tissue-specific isoforms of COX subunit IV, as Northern analysis of muscle, liver, and HeLa cell RNA shows an identically sized transcript in each cell type.

Myosin light chain 1 isoform expression remains constant during ageing in Wistar F455 rats.

In order to study muscle gene expression during ageing, we examined both protein and total cellular RNA from Wistar F455 rat soleus and extensor digitorum longus (EDL) muscles at a variety of chronological ages. We found no evidence of the reappearance of the fast protein isoform of myosin light chain 1 [MLC1] in the slow soleus muscle during ageing previously reported by Syrovy and Gutmann, Pflügers Arch., 369 (1977) 85-89. We used both SDS-PAGE analysis of MLC1 proteins and slot blot RNA analysis with a probe specific for rat fast MLC1 mRNA (pC91), and found no changes in fast MLC1 expression during ageing in soleus or EDL muscles from these rats. These results indicate that re-expression of the fast MLC1 isoform is not a universal property of ageing soleus muscle.

Expression of the GDNF family members and their receptors in the mature rat cochlea.

The GDNF family comprises glial cell line-derived neurotrophic factor (GDNF) and the related proteins neurturin, artemin and persephin, which form a subgroup of the TGF-beta superfamily of growth factors. All four neurotrophic factors provide neuronal cell protection and cell survival. GDNF expression was found in the cochlea, and GDNF has been shown to be effective for inner ear protection from drugs and noise-induced insults. As the other members of the GDNF family also provide protective effects on neuronal cells, they may play important roles in the inner ear. We used RT-PCR to examine the expression of GDNF, neurturin, artemin, persephin and their receptors GFRalpha-1, GFRalpha-2, GFRalpha-3 and c-ret in whole rat cochlea as well as in functionally different subfractions (modiolus and sensorineural epithelium/lateral wall) and compared the levels of neurotrophin and receptor mRNAs in the cochlea to those in substantia nigra brain region. Our results demonstrate the expression of all GDNF family members and their receptors in cochlea and substantia nigra. However, the relative levels of mRNA were different for several genes tested in subfractions of the cochlea and/or compared to expression levels in substantia nigra. The presence of mRNA for all four members of the GDNF family and their preferred receptors in the rat cochlea suggests potential functional importance of these neurotrophic factors as protection and survival factors in the inner ear.

Novel genes expressed in the chick otocyst during development: identification using differential display of RNA.

Differential display of mRNA is a technique that enables the researcher to compare genes expressed in two or more different tissues or in the same tissue or cell under different conditions. The method is based on polymerase chain reaction amplification and comparison of specific subsets of mRNA. We have used this method to clone partial complementary DNAs (cDNAs; amplicons) for genes expressed in the otocyst in order to identify genes that may be involved in development of the inner ear. A full length cDNA was isolated from an embryonic quail head library with an amplicon (KH121) obtained from the otocyst. This avian cDNA encoded a novel, 172-amino acid acidic protein and detected a major transcript of ca 0.8 kb in RNA from chick embryos and several neonatal chick tissues. The full length avian cDNA had high sequence identity to several human cDNAs (expressed sequence tags) from human fetal tissues, including cochlea, brain, liver/spleen and lung, and from placenta. The human homologue of the avian gene encoded a protein that was 183 amino acids long and had 75.6% amino acid sequence identity to the avian protein. These results identified both the avian and human homologues of an evolutionarily conserved gene encoding a small acidic protein of unknown function; however, expression of this gene was not restricted to otocysts.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor complex are expressed in the auditory nerve of the mature rat cochlea.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types which signals through a heterodimer receptor consisting of GDNF-family receptor alpha 1 (GFRalpha-1) and Ret (rearranged during transformation). GDNF expression has previously been reported in the inner hair cells of the rat cochlea, with expression of GFRalpha-1 but not Ret in the cell bodies of the auditory nerve (spiral ganglion cells), using in situ hybridization. The present study used reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry to examine GDNF, GFRalpha-1 and Ret in the adult rat auditory nerve. Semi-quantitative RT-PCR showed expression of GDNF and the two receptor components, GFRalpha-1 and Ret, in the modiolar subfraction of the cochlea containing spiral ganglion cells. A shorter mRNA splice variant for GDNF was also detected. Immunocytochemistry showed immunostaining in the modiolus for GDNF, GFRalpha-1 and Ret that was confined to spiral ganglion cells. When RT-PCR expression levels were compared to the expression in the substantia nigra, GFRalpha-1 expression levels were similar, Ret mRNA was lower in the modiolus and GDNF expression was higher in the modiolus. However, when GDNF was further assessed using Western blot, while GDNF protein was found in the modiolus it was at lower levels than in substantia nigra tissue. These results demonstrate that GDNF and both of its receptor components are found in spiral ganglion cells of the adult rat cochlea. Along with the previous report of GDNF in inner hair cells, these new results provide a basis for the role of GDNF as a survival factor for the auditory nerve, as suggested by previous studies.