Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.     

Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression

We have isolated a chromosomal DNA segment of the human IL-4 gene based on homology with a human IL-4 cDNA sequence and determined its complete nucleotide sequence. The human IL-4 gene, which occurs as a single copy in the haploid genome, is mapped on chromosome 5. It is composed of four exons and three introns and is approximately 10 kilobase pairs in size. 5'-Flanking regions of human and mouse IL-4 genes share about 85% homology extending more than 500 base pairs upstream of a "TATA" like sequence. Several patches of sequences are found in the 5'-flanking region of the human IL-4 gene which are homologous to sequence in the 5'-flanking regions of the IL-2, IL-3, IL-5, and granulocyte-macrophage (GM)-CSF genes. The IL-4 gene is inducible after treatment of human T cell clone by phorbol-12-myristate-13-acetate (TPA) and calcium ionophore A23187. The 2.3-kb 5'-flanking region of the human IL-4 gene transiently transfected into Jurkat human T cell leukemia cells is activated efficiently in response to TPA and A23187 stimulation and, although less efficiently, by human T cell leukemia virus type I-encoded p40x or BPV-encoded E2 protein. Combination of TPA/A23187 and p40x or E2 protein further augmented the level of expression.     

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.     

Multiple mRNAs are generated from the chicken lysozyme gene

We have determined the DNA sequence of a 770 by Pst I fragment containing 450 nucleotides of the 5′ flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5′ end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5′ noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5′ termini of the different mRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5′ ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5′ termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5′ end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.     

Analysis of a drosophila tRNA gene cluster: two tRNALeu genes contain intervening sequences

A recombinant DNA phage containing a cluster of Drosophila melanogaster tRNA genes has been isolated and analyzed. The insert of this phage has been mapped by in situ hybridization to chromosomal region 50AB, a known tRNA site. Nucleotide sequencing of the entire Drosophila tRNA coding region reveals seven tRNA genes spanning 2.5 kb of chromosomal DNA. This cluster is separated from other tRNA regions on the chromosome by at least 2.7 kb on one side, and 9.6 kb on the other. Two tRNA genes are nearly identical and contain intervening sequences of length 38 and 45 bases, respectively, in the anticodon loop. These two genes are assigned to be tRNALeu genes because of significant sequence homology with yeast tRNA3Leu, and secondary structure homology with yeast tRNA3Leu intervening sequence. In addition, an 8 base sequence (AAAAUCUU) is conserved in the same location in the intervening sequences of Drosophila tRNALeu genes and a yeast tRNA3Leu gene. Similar sequenes occur in all other tRNAs containing intervening sequences. The remaining five genes are identical tRNAIle genes, which are also identical to a tRNAIle gene from chromosomal region 42A. The 5' flanking regions are only weakly homologous, but each set of isoacceptors contains short regions of strong homology approximately 20 nucleotides preceding the tRNA coding sequences: GCNTTTTG preceding tRNAIle genes; and GANTTTGG preceding tRNALeu genes. The genes are irregularly distributed on both DNA strands; spacing regions are divergent in sequence and length.