Albumin 3′untranslated region facilitates increased recombinant protein production from Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3′untranslated regions (3′UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3′UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2–3‐fold by replacing the endogenous 3′UTR with the albumin 3′UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3′UTR. Replacement of the native 3′UTR with the albumin 3′UTR led to a 10‐fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3′UTR showed that loss of nucleotides 1–50, which removed an AU‐rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3′UTR sequences.     

The untranslated regions of β-globin mRNA evolve at a functional rate in higher primates

We have sequenced the 3′ and 5′ untranslated regions of β-globin mRNAs from cebus monkey, rhesus monkey and chimpanzee. A comparison with the corresponding human sequences reveals that the rate of sequence divergence among the higher primates is the same in the 3′ and 5′ noncoding regions and that this rate is several times lower than the rate for silent substitutions in the coding regions. In addition, the rate of sequence divergence in the 3′ untranslated region of the primate β-globin mRNA is several times lower than the rate calculated for this region from other comparisons. The low rate of sequence divergence in the noncoding 3′ end of the primate β-globin mRNAs may indicate a specialized and significant function for this region in the higher primates.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Partial sequence analysis of Xenopus alpha- and beta-globin mRNA as determined from recombinant DNA plasmids

Recombinant plasmids containing Xenopus globin mRNA sequences have been constructed using the mRNA:cDNA hybrid conditions of Zain et al. (1979, Cell16, 851–861). The partial nucleotide sequence of two of these recombinants has been determined. They have been identified as containing α- and β-globin-like sequences by homology to other amphibian globin proteins. The nucleotide sequence of these recombinants permits the comparison of conserved regions in both the coding and 3′ nontranslated regions of Xenopus globin mRNAs with the known sequences of other eukaryotic globin proteins and mRNAs. Among the features which have been conserved though evolution is the sequence AAUAAA close to the 3′ terminus of the nontranslated region. Extensive regions of homology occur between the 3′ nontranslated regions of Xenopus α- and β-globin mRNA.     

Selenium-regulated translation control of heterologous gene expression: Normal function of selenocysteine-substituted gene products

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a “selenium translation element” in the 3′ untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3′ untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor β1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile ⁷⁵Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution. © 1996 Wiley-Liss, Inc.     

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.     

Isolation and sequence analysis of a hybrid delta-globin pseudogene from the brown lemur

The β-globin gene cluster of the brown lemur, a prosimian, is very short and contains a single ?-, γ- and β-globin gene, with an additional β-related gene sequence between the γ- and β-globin genes. Brown lemur DNA was cloned into the bacteriophage vector λL47.1 and a recombinant was isolated which contained an 11 × 10³ base insert including the β-globin gene and the additional putative β-globin pseudogene. The nucleotide sequence of this β-related gene was completely determined. A complete gene sequence was found, containing four frameshift mutations sufficient to establish its pseudogene status. The gene was interrupted by two intervening sequences with sizes and locations typical of mammalian β-related globin genes. The pseudogene sequence was compared in detail with human ?-, γ-, δ- and β-globin genes. The beginning of the pseudogene, from the 5′ flanking region to the second exon, was homologous to the corresponding regions of the human ?- and γ-globin genes. In contrast, the second intron, third exon and 3′ flanking region showed a remarkably close homology to the δ-globin, but not β-globin, gene of man. This suggests that the δ-globin gene is not the product of a recent gene duplication, but instead is present in most or all primates. This gene has been silenced on at least two separate occasions in primate evolution (in lemurs and in old world monkeys). In addition, the 5′ end of the lemur ψδ gene appears to have exchanged sequences with an ?- or γ-globin gene, and an analogous exchange with the β-globin gene seems to have occurred recently in the human δ-globin gene. The evolution and function of the δ-globin gene are discussed.     

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with ³²P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.     

Analysis of thyroid hormone effects on myosin heavy chain gene expression in cardiac and soleus muscles using a novel dot-blot mRNA assay

Effects of thyroid hormone on myosin heavy chain (MHC) gene expression were compared in ventricle and soleus muscle of hypothyroid rats, since in this condition, both muscle types express predominately β-MHC mRNA. Changes in MHC mRNAs were analyzed using synthetic oligonucleotide probes complementary to the 3′ untranslated region of the MHC mRNAs. The results indicated that daily treatment with 3,5,3′-triiodo-L-thyronine (T₃) at a dose of 2 μg/100 g body weight increased α-MHC mRNA content in heart muscle by 600% and decreased β-MHC mRNA by 70% within 48 h. In soleus muscle, β-MHC mRNA levels were not affected by 9 wks of treatment, however, Fast IIa-MHC mRNA was increased by 150% at 7 wks and 300% after 9 wks of T₃ administration. Thus, regulation of MHC gene expression by thyroid hormone is both gene and tissue specific.     

Regulation of muscle gene expression: The accumulation of messenger RNAs coding for muscle-specific proteins during myogenesis in a mouse cell line

The expression of RNA sequences coding for myofibrillar proteins has been followed during terminal differentiation in a mouse skeletal muscle cell line. Cloned complementary DNA probes hybridizing with the actins, skeletal muscle α-actin, myosin heavy chain and the myosin alkali light chains were employed in Northern blotting experiments with total cellular poly (A)-containing RNA extracted from the cultures at different times after plating. At the same times, parallel cultures were pulse-labelled with [³⁵S]methionine and the pattern of newly synthesized proteins was analysed by two-dimensional gel electrophoresis. Synthesis of skeletal muscle α-actin and of the myosin alkali light chains (LC_lemb, LC₁, LC₃) was not detectable in dividing myoblast cultures. From the onset of cell fusion, the synthesis of myosin heavy chain, LC_lemb and α-actin increases with similar kinetics. Synthesis of LC₃ (and trace amounts of LC_1F) is detectable and subsequently increases at later stages of myotube formation. The corresponding messenger RNAs coding for myosin heavy chain and skeletal muscle α-actin are first detectable immediately before the initiation of myofibrillar protein synthesis. mRNAs coding for the non-muscle actins are accumulated in myoblasts and diminish after cell fusion. Comparisons between muscle mRNAs depend on the relative sensitivities of the different probes, reflecting mainly their homology with the isoform of the actin or myosin multigene family expressed. Quantitative analysis of Northern blots gives an estimated increase in skeletal muscle α-actin mRNA, with an homologous probe, of at least 130-fold with a minimum level of detection of 40 to 80 molecules per cell. Accumulation of this species and of the myosin heavy chain mRNA follows similar kinetics. mRNA coding for LC₃, the principal myosin light chain detected with the probe, appears to accumulate to a lesser extent initially, paralleling synthesis of the corresponding protein. These results using cloned probes demonstrate a close temporal correlation between muscle mRNA accumulation and protein synthesis during terminal myogenesis in this muscle line.     

Tissue specificity of 3′-untranslated sequence of myosin light chain gene: Unexpected interspecies homology with repetitive DNA

Using the 3′ noncoding and coding sequences of chick heart myosin light chain mRNA cloned into Escherichia coli as probes, it was observed that, while the coding sequence shared homology with myosin light-chain mRNAs from other sources, the 3′ noncoding sequence was specific for chick heart muscle. This property was used to detect chick heart-specific myosin light-chain gene activity in chick blastoderms of very early developmental stages where cells of different muscle origins cannot be distinguished morphologically. However, in spite of the tissue-specific divergence of the 3′ noncoding sequence of myosin light-chain gene, which is present in a single copy in the chick genome, a surprising homology with DNA from such a diverse source like Dictyostelium discoideum was noted. The sequence homologous to chick myosin light-chain DNA was apparently present in a high repetition frequency in the Dictyostelium genome.