Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken

Summary The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A+T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.     

AT-rich linkers in long range organisation of Drosophila DNA

Summary AT-rich segments were mapped in 6 different domains of the Drosophila melanogaster genome by partial denaturation of cloned genomic DNA segments and observation in the electron microscope (EM). As found in a previous investigation (Moreau et al., 1982), three types of AT-rich segments could be distinguished: 1) AT-rich linkers with a very high AT content, 600±200 bp long; 2) clusters of such AT-linkers extending over up to 10 Kpb and 3) AT-rich stretches which are shorter and of lower AT content compared to AT-linkers. Six genes previously localized in these domains were found to lie in relatively GC-rich segments framed by AT-rich elements of the 3 types: the Larval Serum Protein LSP-1 gene is framed by 2 AT-linkers, the LSP-1 and LSP-2 genes by two AT-rich stretches, and the LSP-1 gene by a cluster and a stretch. The 55 Kbp genomic segment encompassing the P6 gene contains and AT-cluster of about 15 Kbp and several AT-linkers and AT-rich stretches. The 85 Kbp domain containing the P1 gene includes 3 AT-rich clusters of about 10 Kbp each framing GC-rich domains punctuated by AT-linkers and stretches. This study shows that AT-mapping allows a rapid diagnosis of large genomic DNA domains in relation to the AT-rich segments which, possibly, are of significance with regard to genome organisation and function.     

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Evolution of the human sarcomeric-actin genes: Evidence for units of selection within the 3′ untranslated regions of the mRNAs

Summary The complete 3 untranslated region (3UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (T_m) measurements, we found that the cardiac-actin gene 3UTR also consists of conserved and nonconserved segments. Comparison of human andXenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3UTR. We conclude that subsegments of the 3UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.     

Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples

Summary 3-Aminoglycoside phosphotransferase [APH(3)] enzymes are a group responsible for resistance to the antibiotics kanamycin (Km) and neomycin (Nm) in bacteria. Escherichia coli ECT24, originally isolated from a meat sample, harboured an 83-kb conjugative R-plasmid (pRPJ24) that carries transferable resistance to Km and Nm. Plasmid pRPJ24 was transferred by conjugation to Enterobacter cloacae 94R, which was used as the source of plasmid DNA in development of a probe for the Km-resistance determinant. Random cloning of BamHI and HindIII double-digest restriction fragments of pRPJ24 in the pUC18 vector plasmid produced clones resistant to both Nm and Km carrying a 1.9-kb DNA insert. Southern hybridization of pRPJ24 cloned chimeric plasmid DNA (pKPJ94) showed homology with the APH(3)II gene from transposon Tn5. A PstI digest of pKPJ94 produced a 920-bp fragment which hybridized with the APH(3)II structural gene, and was used as a DNA probe for the APH(3)II subclass gene. A 980-bp BamHI fragment from plasmid pGH54 carrying the APH(3)I gene from transposon Tn903 was used as a subclass I probe. Total DNA from 206 randomly screened Km-resistant Enterobacteriaceae isolates from raw ground beef and chicken meat samples were examined for the occurrence of APH(3) subclass I and II using non-radioactively-labelled DNA probes. Thirty-six percent and 60% of the isolates examined carried subclass I and II resistances, respectively, in the isolates from chicken meat samples. The corresponding values for bacterial strains from raw ground beef samples were 51% and 72%, respectively. Four percent of the resistant bacterial isolates from chicken samples did not display homology to either probe. This value was 28% for bacterial isolates from ground beef. Three percent of bacterial isolates from chicken samples and 44% from ground beef samples displayed homology to both APH(3) I and II DNA probes. Offprint requests to: A. H. G. P. Jayaratne     

Characterization of genomic DNA of mycobacteriophage I3

The GC content of mycobacteriophage I3 DNA is 67% as determined from thermal melting analysis, buoyant density in CsCl gradient, and 5-deoxymononucleotide analysis. High-resolution melting of I3 DNA revealed that the base distribution is random. Studies with methylation-specific restriction enzymes and high voltage electrophoretic analysis of 5-deoxymononucleotides did not indicate the presence of any unusual or methylated bases in I3 DNA. The molecular size of I3 DNA is estimated to be about 135 Kbp, based on the restriction fragment size distributions and sedimentation in sucrose gradients. The restriction cleavage pattern by a variety of restriction endonucleases has been determined. The circularly permuted nature of I3 DNA, indicated from the restriction patterns, has been confirmed by Southern blot hybridizations and 5 end group analysis.     

Restriction patterns of adult chicken globin genes at early and late stages of embryonic development

Globin mRNA isolated from anemic chicken was transcribed into cDNA and integrated into the Pst I cleavage site of plasmid pBR 322. After cloning in E. coli strain HB 101 and colony hybridization with¹²⁵I-labelled globin mRNA the plasmids of individual clones were characterized by hybrid arrested cell-free translation. Thus we could isolate clones containing or globin chain nucleotide sequences.DNA was isolated from chicken blastoderms incubated for 18–20 h and from 11 d chicken embryos. A comparison of the restriction maps of the DNA from the two developmental stages with labelled nick translated plasmids and labelled cDNA did not indicate any globin gene rearrangements between these two stages of embryonic development. We conclude, that the adult chicken globin genes show a constant genomic organization during embryonic development. However, the restriction patterns of the globin gene family of the chicken strain investigated revealed some differences after 2 generations of propagation.     

Cloning of the fibroin gene from the oak silkworm, Antheraea yamamai and its complete sequence

The nucleotide sequences containing an entire genomic region and 5 upstream region of Antheraea yamamai fibroin gene have been determined. The gene consists of an initial exon encoding 14 amino acids, an intron (150 bp), and a long second exon coding for 2641 amino acids. The fibroin coding sequence shows a specialized organization with a highly repetitive region flanked by non repetitive 5 and 3 ends. Northern blot analyses confirmed that fibroin gene is actively expressed in the posterior silk gland of the final instar larvae of Antheraea yamamai.     

Den 1, Den2 and Den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5-ends of single-stranded DNA or double-stranded DNA with either blunt or 5-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3-exonuclease with double-stranded DNA possessing either blunt or 3-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3-5 direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.     

Structural and functional properties of DNA polymerase delta from rabbit bone marrow

Summary DNA polymerase delta, the most recently described class of eukaryotic DNA polymerase, has been purified to apparent homogeneity from rabbit bone marrow. Unlike the previously known eukaryotic DNA polymerases, delta has a 3 to 5 exonuclease as an integral component of its 122 000 molecular weight, single polypeptide structure. Similar to the function with prokaryotic DNA polymerases, the 3 to 5 exonuclease assists DNA polymerase delta in maintaining the fidelity of DNA synthesis by excising misincorporated nucleotides. DNA polymerase delta and the longer known eukaryotic DNA polymerase alpha are similar in many features. Both are very sensitive to sulfhydryl inhibitors such as N-ethylmaliemide (NEM) and to the antibiotic aphidicolin. Such criteria distinguish alpha and delta from DNA polymerases beta and gamma. This has led to the conclusion that nuclear DNA replication, which is sensitive to NEM and aphidicolin, is carried out by DNA polymerase alpha. However, the similar sensitivity of delta to these reagents requires that the role of alpha and delta in nuclear DNA replication be further defined. In many features DNA polymerase delta is also similar to the viral induced DNA polymerases such as the Herpes simplex virus DNA polymerases which also have associated 3 to 5 exonuclease. Understanding of DNA synthesis and the mechanism of DNA replication fidelity in mammalian cells depends upon a further understanding of both DNA polymerases alpha and delta and the nature of the relationship they have to each other.     

The chromosomal distribution of repetitive DNA sequences within the human β globin gene cluster

Summary The chromosomal distribution and degree of repetitiveness of the sequences within the human globin gene cluster has been studied by in situ hybridisation. A genomic recombinant, H\G1, which contains 15.9 thousand base pairs (kb) of DNA inserted into charon 4A, was used as a template for the [³H] complementary RNA used for hybridisation. The inserted DNA contains sequences 4.7 kb to the 5 side of the gene and continues through the and genes to a site 2.8 kb to the 3 side of the gene. Two highly repetitive sequences, which are distributed evenly over all the chromosomes, have been identified within this DNA. Another somewhat less repetitive sequence has been identified between the and genes.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat

Summary The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and -fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and -fetoprotein genomic subclones with ³²P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG·dT repeats by using ³²P-labeled poly-(dG·dT)(dC·dA) oligomer probe. They occurred in 5 and 3 extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element, 4D12. The intense repetitive sequence, localized in the 3 non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the -fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.     

Probing surface structure of sarcin domain on ribosomes ofEscherichia coli by complementary oligo DNAs and ribosome-inactivating protein

The regional structure of the sarcin domain of 23S rRNA ofEscherichia coli ribosomes was determined by a combinatory approach of oligo DNA probes and the action of -sarcin. The sarcin domain is protected by a reactive complementary oligo DNA probe against the hydrolytic action of -sarcin. This protective effect is dependent upon the length and the complementary sequence of oligo DNA probes that react to ribosomes. Under UV irradiation and using of the primer extension, nucleotides that contacted by reactive oligo DNA probes were determined. Nucleotides at the 3 side of the domain (positions from G2659 to C2676) were targeted by oligo DNA probes that have their sequences to complement the domain, indicating that the 3 side region was exposed on the surface of ribosomes, whereas nucleotides at the 5 side of stem and extented to two bases at the loop (positions from C2646 to A2654) were not accessible to any oligo DNA probes, implying that the region could be buried in ribosomes. This study also provided evidence that the conformation of the sarcin domain is subjected to alteration if the exposed 3 side of domain is targeted by the reactive DNA probe. The importance of the topological arrangement of the sarcin domain that engages in the translocation event during translation is discussed.     

Repeated sequences from theArabidopsis thaliana genome function as enhancers in transgenic tobacco

Sixteen segments ofArabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation ofNicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenicN. tabacum. The sequences were average with respect to the AT content ofA. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5-CCTCT-3 and 5-AAGGAT-3). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.     

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3 half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3 half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800–1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.