Wayne Connor

Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

SummaryTwelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.

An H1 histone gene from rainbow trout (Salmo gairdnerii)

SummaryA 1.7-kbp DNA region from the 10.2-kb cluster containing the five rainbow trout histone genes has been subcloned in pBR322 and completely sequenced. It contains a trout histone H1 gene together with its 5′ and 3′ flanking sequences. This H1 gene codes for a H1 variant different from the major trout testis H1 previously sequenced by Macleod et al. (1977). Northern blots of total RNA from trout testis, kidney, and liver indicate that this H1 gene is expressed in all three tissues but that the level of H1 mRNA is much higher in testis than in other tissues. The lack of heterogeneity in the sizes and 5′ initiation sites of trout H1 mRNAs is surprising in view of the substantial heterogeneity of H1 variant proteins observed previously. The coding sequence of the H1 gene shows strong evidence of repeated partial duplications of a hexapeptide motif of the form Ala.Ala.Ala.Lys.Lys.Pro and of a pentapeptide phosphorylation-site sequence, Lys.Ser.Pro.Lys.Lys, during its evolution. Comparisons are drawn between this gene and the coding sequences of other vertebrate H1 genes from chicken andXenopus, and a strong homology is seen in the region of amino acids 22–101, which form the hydrophobic “head” of the H1 molecule. The 5′ and 3′ regulatory signals in the trout H1 are also compared with those of H1 genes from other sequences.

Organization and nucleotide sequence of rainbow trout histone H2A and H3 genes

SummaryA 2.56-kbp fragment containing genes coding for histones H2A and H3 that forms a portion of the 10.2-kbp cluster containing all five histone genes isolated from a λ-Charon 4A library of rainbow trout genomic DNA has been characterized in detail and its complete nucleotide sequence determined. The genes are arranged in tandem, being encoded on the same DNA strand. They are separated by 380 bp of intergenic spacer DNA that contains an alternating purine-pyrimidine stretch of 20 bp and a 46-bp stretch that has the potential of forming a triple cruciform structure. The histone genes contain no introns, have the RNA polymerase II promoter-associated signals known as CAAT and TATA boxes in their 5′ flanking regions and contain a conserved inverted repeat sequence, similar to that found in histone genes of other species, capable of forming a hairpin structure at the 3′ end of the transcription unit.

Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii).

SummaryThe complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone λTH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5′ and 3′ flanking and spacer sequences, and represents the 5′ half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5′ and 3′ ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure.

Oligonucleotides as probes for mammalian protamine mRNAs

Protamine-like sequences have been identified in poly A(+ ) mRNAs from mammalian testes by the use of a common, complementary oligonucleotide (GCAGCANCK PTANCKNGCCAT; predicted from the common N-terminal amino acid sequence, MARYRCC, seen in several mammalian P1 protamines [D. J. McKay, B. S. Renaux and G. H. Dixon,Bioscience Reports5:383–391 (1985)]). This oligonucleotide was utilized to prepare species-specific, primer-extended transcripts for use as Northern blotting probes. Analysis of the mRNA primer-extended transcripts revealed a discrete and similar set of products common to both bull and rat testis mRNAs which were distinct from those obtained from human testis mRNA. Northern analysis of total poly A(+) mRNAs from the corresponding mammalian testis was consistent with these results and suggests that bull and rat protamine mRNAs are more closely related to each other than to human protamine mRNA.

Restriction fragment length polymorphism (RFLP) analysis of bovine nuclear protein genes

SummaryWe have recently cloned both the bovine protamine (Krawetz et al. 1987, DNA 6: 47–57) and high mobility group (HMG-1) cDNAs (Pentecost and Dixon 1984, Bioscience Reports 4: 49–57). They have been used as probes for Restriction Fragment Length Polymorphism analysis of male-female pairs of different species and breeds, within the genus Bos. Utilizing this approach we have studied inheritance, chromosomal location and gene copy number of the bovine protamine and HMG-1 genes. This revealed that these nuclear protein genes are highly conserved suggesting that selective pressure has maintained their gene structures during evolution. A polymorphic Taq 1 restriction fragment was identified that was shown to be a heritable marker. These genes are not sex-linked and are present in a single copy for protamine and at least two copies for the HMG-1.